Expression, purification and uses of a Plasmodium falciparum liver stage antigen 1 polypeptide

ABSTRACT

In this application is described the expression and purification of a recombinant  Plasmodium falciparum  (3D7) LSA-NRC polypeptide. The method of the present invention produces a highly purified polypeptide which is useful as a vaccine and as a diagnostic reagent.

This application claims the benefit for priority under 35 U.S.C. §119(e)from Provisional Application Ser. No. 60/425,719 filed on Nov. 12, 2002.

INTRODUCTION

Plasmodium falciparum is the leading cause of malaria morbidity andmortality. The World Health Organization estimates that approximately200 million cases of malaria are reported yearly, with 3 million deaths(World Health Organization, 1997, Wkly. Epidemiol. Rec. 72:269-276).Although, in the past, efforts have been made to develop effectivecontrols against the mosquito vector using aggressive applications ofpesticides, these efforts ultimately led to the development of pesticideresistance. Similarly, efforts at treatment of the disease throughanti-parasitic drugs led to parasite drug-resistance. As the anti-vectorand anti-parasite approaches failed, efforts became focused on malariavaccine development as an effective and inexpensive alternativeapproach.

However, the complex parasitic life cycle has further confounded theefforts to develop efficacious vaccines for malaria. The parasite's lifecycle is divided between the mosquito-insect host and the human host.While in the human host, it passes through several developmental stagesin different organellar environments, i.e. the liver stage and the redblood stage. Although conceptually simple, in reality the problems thatmust be considered when designing subunit vaccines for malaria aregreat. Antigen diversity is a characteristic that must be taken intoaccount and includes a high degree of developmental stage specificity,antigenic variation and antigen polymorphism.

The observation that sterile immunity to malaria can be induced byimmunization with irradiated sporozoites (Clyde et al., 1973.J. Med.Sci. 266: 169-277; Krzych et al. 1995, J. Immunol. 155, 4072-4077;Hoffman et al., 2002 JID 185, 1155-1164) has focused attention on thesporozoite and liver stages of the parasite life-cycle as potentialtargets of an effective vaccine. Liver stage antigen 1 (LSA-1) is aprime candidate for development as a vaccine as it is expressed duringthe hepatic stage of infection. It is known that peptides or recombinantfragments of the LSA-1 protein elicit specific humoral, cellular andcytokine immune responses from cultured peripheral blood mononuclearcells (PBMC) taken from malaria exposed individuals. These immuneresponses are correlated with a reduction or absence of parasitemia andfalciparum malaria disease in subsequent transmission seasons (Kurtis etal. 2001, Trends in Parasitology. 17, 219-223). The P. falciparum LSA-1protein is found within the parasitophorous vacuole (PV), a spacedefined as that region between the inner plasmalemma and the outerparasitophorous vacuole membrane (PVM). The (PV)forms a distinct ringseparating the parasite cytoplasm from the host hepatocyte (Fidock etal., 1994, J. Immunol. 153, 190-204). The PVM is of host origin and isformed by invagination of the host cellular membrane when the parasiteinvaded the host cell. The LSA-1 protein is approximately 230 kDa inmass. Its expression begins shortly after sporozoite invasion of theliver and increases with liver stage development. It is described as aflocculent material within the parasitophorous vacuole and may alsoadhere to the surface of merozoites, suggesting a crucial role in liverschizogony perhaps protecting the merozoite surface (Hollingdale et al.,1990, Immunol. Lett. 25, 71-76). When the hepatocyte ruptures, itreleases the merozoites encased in LSA-1 protein into the liver sinusoidand into the blood stream (Terzakis et al., 1979, J. Protozool. 26,385-389).

The LSA-1 protein is characterized by a large central repeat regionconsisting of about eighty-six 17 amino acid tandem repeats flanked byshort non-repetitive N-terminal and C-terminal regions which are highlyconserved across strains. Studies have revealed the protein is a targetof B-cells (Guerin-Marchand et al. 1987, Nature 329,164-167; Fidock, etal., 1994, supra; Luty et al., 1998, Eur. Cytokine Netw. 9, 639-646),helper T-cells (Doolan and Hoffman, 2000, J. Immun. 165, 1453-1462;Fidock et al. 1994, supra; Connelly et al., 1997, Infect. Immun. 65,5082-5087, Luty et al., 1998, supra; Kurtis et al., 1999, supra) andMHC-restricted CD8+ CTLs (Hill et al., 1991, Nature 532, 595-600; Aidooet al., 1995; Doolan and Hoffman, 2000, J. Immunol. 165, 1453-1462).T-cell epitopes have been defined amongst the amino acid residues in theN-terminal and C-terminal flanking regions and in the central repeatregion (Doolan and Hoffman, 2000, J. Immun. 165, 1453-1462, 2000; Kryzchet al., 1995, J. Immunol. 155, 4072-4077; Hill et al., 1991, supra;Fidock et al., 1994, supra). Even though LSA-1 is one of the mostimmuno-epidemilogically studied P. falciparum malaria antigens a vaccineusing the protein has not yet been developed.

The P. falciparum lsa-1 gene sequences have been used for over a decadein an attempt to make a DNA vaccine against P. falciparum. NYVAC-Pf7, amultivalent poxvirus vector made by WRAIR and Virogenetics, contained anlsa-1 gene that encoded a repeatless protein. The NMRC MuStDO5 (amixture of DNA plasmids constructs encoding part or all of five P.falciparum genes: CS, SSP2, LSA-1, EBA-175 and MSP-1), and more recentlyCSLAM (a mixture of DNA plasmid vaccine constructs encoding all or partsof five P. falciparum malaria genes: CS, SSP2, LSA-1, AMA-1 and MSP-1)contain LSA-1 gene sequences in their vaccines. The Oxford Universityscientists have modified vaccinia Ankara (MVA) and cowpox constructscontaining DNA sequences that code for several LSA-1 T-cell epitopes.NMRC is currently constructing alpha-virus (VEE replicons) andadenovirus constructs that contain lsa-1 genes.

All these potential vaccines use LSA-1 gene constructs designed asinjectable DNA sequences that will be transcribed and translated in thehuman host, for example in a DNA plasmid, poxvirus, adenovirus, etc. Theresearchers have used LSA-1 DNA sequences that express the protein inthe immunized host rather then injection of isolated LSA-1 protein sbecause the LSA-1 protein has proven to be impossible to obtain. It hasbeen very difficult or impossible to express in bacteria, yeast orbaculovirus. Therefore recombinant expression and isolation of theprotein at a scale that is commercially viable for vaccine developmentand use has never been achieved.

We have overcome this problem and now can express the protein inbacteria. The expressed product can be isolated and purified to highhomogeneity for use as an immunogen or a vaccine.

SUMMARY OF THE INVENTION

The aim of the present invention is to develop a P. falciparumliver-stage directed vaccine that will result in lower parasite burdenin the human host. To that aim, large-scale expression, purification andcharacterization of a P. falciparum LSA-1 (PfLSA1) immunogenic peptideis necessary. The LSA-1 native protein is about 230 kDa in size andcontains a large central segment made up of highly conserved tandem 17amino acid repeat units. The 3D7 clone of P. falciparum contains 86repeats. The amino acid sequences of the N-terminal and C-terminalportions of the molecule are highly conserved in different isolatesexamined (Fidock, et al, 1994, supra).

We have designed an LSA-1 polypeptide, LSA-NRC, containing a number ofT-cell and B-cell epitopes. Our gene construct contains codons for onlythe N-terminal (#28-154 residues), the C-terminal (#1630-1909 residues)and two 17 amino acid residue repeats (residue numbers refer to Genbankprotein sequence for P. falciparum, ID # A45592). We chose to includeone copy of the major 17 amino acid repeat,GluGlnGlnSerAspLeuGluGlnGluArgLeuAlaLysGluLysLeuGln (SEQ ID NO:1) thatoccurs 31 times in the P. falciparum 3D7 clone LSA-1 protein, and onecopy of a minor repeatGluGlnGlnArgAspLeuGluGlnGluArgLeuAlaLysGluLysLeuGln (SEQ ID NO:2) thatoccurs 4 times in 3D7 clone protein. These regions have been found tocontain T-cell epitopes, including those identified as being associatedwith immune responses in man (T1, J, LSA-Ter, 1s6, T3, T5, ls8), andseveral B-cell epitopes, including the two central repeat regions.

The extreme AT richness of the P. falciparum lsa-1 gene makesover-expression in most cell types other than the parasite precariousdue to the unusual codon usage. Two codon modification approaches wereundertaken to improve the protein yield by genetically re-engineeringthe gene sequence to introduce nonsynonymous mutations (changes in thenucleic acid sequence that still encode the same amino acid) in order toimprove translation rates.

In the first approach, the constructs were designed by substitutingfrequently used E. coli synonymous codons, for the infrequently usedcodons specified by the P. falciparum gene, referred to as ‘codonoptimization’. In this approach, the same E. coli codon is used everytime a given amino acid is specified (e.g. CGG for every arginine).

In the second approach, the constructs were designed to “harmonize”translation rates, as predicted by comparison of codon frequency tablesbetween P. falciparum and E. coli, in an attempt to mimic nativetranslation and proper protein folding. More specifically, the frequencyof occurance of each codon in the P. falciparum gene was calculated andreplaced with an E. coli codon with a similar frequency for the sameamino acid. Please see U.S. application of Angov et al., Ser. No.10/440,668, filed on 1 Apr. 2003, for more information on“harmonization”. An example of each approach is shown in Table 1. TABLE1 Original Usage rate of E. coli Codon Usage Harmonized Codon Usage P.falciparum original codons abundance rate of lsa-nrc^(e) lsa-nrc^(h)rate of lsa-nrc^(h) Codons in P. falciparum optimized in E. coli codonsin E. coli AAC 0.14 AAC 0.94 AAT 0.06 TTG 0.14 CTG 0.83 CTC 0.07 AGA0.59 CGT 0.74 CGC 0.25

Two constructs were designed with these approaches in mind. A geneconstruct, lsa-nrc^(e), was designed based on the optimized codon usageof the highly expressed genes in E. coli cells. Transforming various E.coli cell lines with this construct allowed expression of solubleprotein. However, cryopreserved, transformed cells were geneticallyunstable and failed to express LSA-NRC(E) protein aftercryopreservation. Additionally, induction of expression of LSA-NRC(E)polypeptide resulted in complete plasmid loss and cell death. Theapparent toxicity associated with lsa-nrc^(e) gene expression and ourinability to store transformed cells, made these constructs impracticalfor vaccine production.

In the second approach, a DNA gene construct, lsa-nrc^(h) was designedby harmonized codon usage. Transforming various E. coli cell lines withthis plasmid allowed expression of a soluble protein, and cyropreservedtransformed cells were genetically stable and expressed the recombinantLSA-NRC(H) polypeptide. Sequencing of the resulting protein orpolypeptide revealed that an insertion of one amino acid was present inthe T5 epitope of the recombinant protein. This protein was thenreferred to as LSA-NRC(H)Mut and is described in the Examples below. Weare presently removing the inserted amino acid in order to express aLSA-NRC(H) polypeptide product without the insertion.

We have developed a three column chromatographic purification schemethat results in an LSA-NRC(H) product that is >99% pure. Briefly, thebacterial cells are cracked with a mircrofluidizer and incubated withlow levels of sarkosyl, a detergent to facilitate the removal ofendotoxin. The lysate is passed over Ni-NTA and bound material is washedwith a buffer with high salt and low pH before elution with imidazole.The product is further purified using DEAE and SP-Sepharose ion exchangechromatographic procedures. The final amount of purified proteinobtained is approximately 5 g/Kg of starting bacterial paste.

Balb/c mice have been immunized with LSA-NRC(H)Mut polypeptideemulsified in Montanide ISA-720 adjuvant. The mice responded to theprotein by making IgG antibodies indicating that the polypeptide wasimmunogenic (FIG. 4). These antibodies recognize in vitro cultured livercells infected with P. falciparum 3D7 parasites (FIG. 8) indicating thatthe antibodies made can recognize the LSA-1 protein in its nativestructure.

Therefore, it is an object of the present invention to provide arecombinant P. falciparum LSA-NRC polypeptide for stimulation ofantibody production and T-cell immune responses upon vaccination ofindividuals and for use in diagnostic assays. When expressed in a host,the P. falciparum codon usage is preferably harmonized to the host codonusage, at which point, the expressed product is referred to as anLSA-NRC(H) polypeptide. The exemplified LSA-NRC(H)Mut recombinantpolypeptide is harmonized for expression in E. coli and consists of theharmonized N-terminal, C-terminal and two tandem 17 amino acid repeatsof the LSA-1 protein of P. falciparum in addition to an amino acidinsertion in the T5 P. falciparum epitope. The LSA-NRC(H)Mut polypeptidealso includes a 6×His tag on the C-terminal end of the polypeptide foraid in purification of the polypeptide. The nucleotide sequence of thelsa-nrc^(hmut) is specified in SEQ ID NO:3 and the encoded amino acidsequence is specified in SEQ ID NO:4.

Variations of LSA-NRC include peptides with repeats from 0 to 90 whereinthe repeats contain conserved amino acids of the same basic 17 aminoacids following the order: X ¹ GlnGlnX ² AspX ³ GluGlnX ⁴ ArgX ⁵ AlaX ⁶GluX ⁷ LeuGln (SEQ ID NO:5) where x₁ is either Glu or Gly; x₂ is Ser orArg; x₃ is Asp or Ser; x₄ is Glu or Asp; x₅ is Leu or Arg; x₆ is Lys orAsn and x₇ is Lys or Thr or Arg. The repeat unit of 17 amino acids canstart at any of the amino acids in the 17 amino acid unit, much like thestart of a circle can have many points. However, because of thealpha-helical, coiled-coil nature of the tertiary structure of therepeats each unit should maintain the basic ordering of the amino acids.

Similarly, LSA-NRC peptides could also contain less or more of theN-terminal region of LSA-1, extending from the first methionine atresidue #1 to around residue #158, and/or more or less of the C-terminalregion of LSA-1, extending from approximately amino acid residue #1630-1909, keeping in mind that these regions contain T-cell epitopeswhich aid in mounting an immune response. The defined epitopes in theseregions, shown in Table 2, can be included or excluded, renderedfunctional, i.e. recognized by an epitope defining-antibody, ornonfunctional, not recognized by an epitope-defining antibody, dependingon the intended purpose of the resulting expressed peptide orpolypeptide. The N- and C-terminal regions can additionally bepresented, whether complete or partial, in multiples of 1 in order toachieve a desired immune response. In addition, insertions, deletions orsubstitutions can be designed to enhance immunogenicity or reduceimmunogenicity of one or more epitopes. As is described in the Examplesbelow, we have designed LSA-NRC(H)Mut wherein the T5 epitope isinterrupted by an insertion of one amino acid, an arginine (please seeTable 2, SEQ ID NO:15 for the amino acid sequence of the mutant T5epitope) and have found that the resulting polypeptide is immunogenic.TABLE 2 Most Common Studied Epitopes of P. falciparum LSA-1. InclusiveAmino Epitope Acid Name Numbers Sequence References T1  84-107LeuThrMetSerAsnValLysAsnValSerGlnThr Krzych,AsnPheLysSerLeuLeuArgAsnLeuGlyValSer 1995; (SEQ ID NO:6) Connelly, 1997LSA-Rep — GluGlnGlnSerAspLeuGluGlnGluArgLeuAla Fidock, LysGluLysLeuGln(SEQ ID NO:7) 1994; Jurgen 2001 J 1613-1636GluArgLeuAlaLysGluLysLeuGlnGluGlnGln Fidock, ArgAspLeuGluGln (SEQ IDNO:8) 1994; Jurgen 2001 NR 1633-1659ThrLysLysAsnLeuGluArgLysLysGluHisGly Fidock, AspValLeuAlaGluAspLeuTyr1994; (SEQ ID NO: 9) Jurgen 2001 LSA-Ter 1686-1719AsnSerArgAspSerLysGluIleSerIleIleGlu Fidock,LysThrAsnArgGluSerIleThrThrAsnValGlu 1994;GlyArgArgAspleuHisLysGlyHisLeu Jurgen 2001 (SEQ ID NO:10) 1s6 1786-1794LysProIleValGlnTyrAspAsnPhe Hill, 1992 (SEQ ID NO:11) T5 1813-1835AsnGluAsnLeuAspAspLeuAspGluGlyIleGlu Krzych,LysSerSerGluGluLeuSerGluGluLysIle 1995; (SEQ ID NO:12) Connelly, 1997ls8 1850-1856 LysProAsnAspLysSerLeu (SEQ ID NO:13) Hill, 1992 T51888-1909 AspAsnGluIleLeuGlnIleValAspGluLeuSer Krzych,GluAspIleThrLysTyrPheMetLysLeu 1995; (SEQ ID NO:14) Connelly, 1997T5-MUtR 1888-1909 AspAsnGluIleLeuGlnIleValAspGluArgLeu Hillier,SerGlueAspeIleThrLysTyrPheMetLysLeu unpublished (SEQ ID NO:15) LSA1.1 84-104 LeuThrMetSerAsnValLysAsnValSerGlnThr JoShi, 2000AsnPheLysSerLeuLeuArgAsnLeuGlyValSer (SEQ ID NO:16) LSA1.2 1742-1760HisThrLeuGluThrValAsnhleSerAspValAsn Joshi, 2000AspPheGlnhleSerLysTyrGlu (SEQ ID NO:17) LSA1.3 1779-1796AspGluAspLeuAspGluPheLysProIleValGln Joshi, 2000 TyrAspAsnPheGlnAsp (SEQID NO:18) LSA1.4 1797-1816 IleGlyIleTyrLysGluLeuGluAspLeuIleGlu Joshi,2000 Lys (SEQ ID NO:19) LSA1.6 1817-1834AsnGluAsnLeuAspAspLeuAspGluGlyIleGlu Joshi, 2000LysSerSerGluGluLeuSerGluGluLysIle (SEQ ID NO:20) LSA1.6 1835-1849IleLysLysGlyLysLysTyrGluLysThrLysAsp Joshi, 2000 AsnAsnPhe (SEQ IDNO:21) LSA1.7 1888-1909 AspAsnGluIleLeuGlnIleValAspGluLeuSer Joshi, 2000GluAspIleThrLysTyrPheMetLysLeu (SEQ ID NO:22) Doolan 1671-1679TyrTyrIleProHisGlnSerSerLeu Doolan, 1671 (SEQ ID NO:23) unpublishedKrzych et al., 1995. J. Immunol. 155, 4072-4077 Fidock et al., 1994. J.Immunol. 153, 190-20 Hill, et al, 1992. Nature 360, 434-439 Joshi, etal., 2000. Infect Immun. 68, 141-150 Connally et al., 1997. InfectImmun. 65, 5082-5087 Jurgen, et al. , 2001. JID. 183, 168-172

The LSA-NRC polypeptide could optionally include the signal sequencecontained in the first 28 residues of the native protein, and/or anyother amino acid sequence found in other strains or clones of the P.falciparum lsa-1 gene.

When designing the LSA-NRC polypeptide, the epitopes or fragments of thelsa-1 gene chosen may be linked in tandem, N-terminal to C-terminal, inrandom or fixed order by their ends or cross linked by chemical means bytheir amino acids.

It is another object of the present invention to provide compositionscomprising purified recombinant P. falciparum LSA-NRC polypeptide.

It is yet another object of the present invention to provide novelvector constructs for recombinantly expressing P. falciparum lsa-nrc, aswell as host cells transformed with said vector.

It is also an object of the present invention to provide a method forproducing and purifying recombinant P. falciparum LSA-NRC polypeptidecomprising:

growing a host cell containing a vector expressing P. falciparum LSA-NRCpolypeptides in a suitable culture medium,

causing expression of said vector sequence as defined above undersuitable conditions for production of soluble polypeptide and,

lysing said transformed host cells and recovering said LSA-NRCpolypeptide such that it is essentially free of host toxins.

It is also an object of the present invention to provide diagnostic andimmunogenic uses of the recombinant P. falciparum LSA-NRC polypeptide ofthe present invention, as well as to provide kits for diagnostic assaysfor example in malaria screening and confirmatory antibody tests.

It is also an object of the present invention to provide monoclonal orpolyclonal antibodies directed against LSA-NRC which also react withLSA-1, more particularly human monoclonal antibodies or mouse monoclonalantibodies which are humanized, which react specifically with nativeLSA-1 epitopes, either comprised in peptides or conformational epitopespresent in the native parasite or in recombinantly expressed proteins.

It is also an object of the present invention to provide possible usesof anti-LSA-NRC monoclonal antibodies for malaria antigen detection orfor therapy to prevent malaria reinfection.

It is yet another object of the present invention to provide a malariavaccine or an immunogenic composition comprising LSA-NRC of the presentinvention, in an amount effective to elicit an immune response in ananimal or human against P. falciparum; and a pharmaceutically acceptablediluent, carrier, or excipient.

It is another object of the present invention to provide a method foreliciting in a subject an immune response against malaria, the methodcomprising administering to a subject a composition comprising LSA-NRCof the present invention.

The vaccine according to the present invention is inherently safe, isnot painful to administer, and should not result in adverse side effectsto the vaccinated individual.

All the objects of the present invention are considered to have been metby the embodiments as set out below.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1. Protein gel showing purification of LSA-NRC(H)Mut after threecolumn chromatographic purification.

FIG. 2. Purification profiles of LSA-NRC(H)Mut protein after each of thechromatography steps used in purification. The profile is a chartrecording tracing of the OD absorbance of the column effluent at 280 nm.

FIG. 3. Silver stained SDS-PAGE gel of final purification product afterelution off SP-Sepharose.

FIG. 4. Development of antibody in two strains of mice, Balb/c andC57BL/6, after immunization with LSA-NRC(H)Mut in Montanide ISA-720 bythe subcutaneous route of injection. Each symbol represents the titer ofone mouse in that group. P-1, P2 and P3 refer to samples of sera takentwo weeks post-1, post-2 and post-3 after immunization with theindicated amount of protein. Balb/C were responders to the polypeptidewhile c57Bl/6 were generally non-responders.

FIG. 5. Isotype of antibody molecules from responder Balb/c mice toLSA-NRC(H)Mut. The major isotype antibody formed in Balb/c mice wasIgG1, with some IgG2a and IgG2b formed. Very little IgG3 was made inBalb/C mice in response to LSA-NRC(H)Mut.

FIG. 6. Interferon-gamma production in C57Bl/6 mice. Spleen cells frommice immunized with LSA-NRC(H)Mut tended to produce IFN-gamma asdetected by ELISPOT assay.

FIG. 7. Rabbits immunized with LSA-NRC(H)Mut make antibodies thatrecognize the N-terminal and the C-terminal protions of LSA-NRC^(MutR).LSA-1N+trx and LSA-1C+trx are bacterial expressed domains of LSA-1(Kurtis, et al 2001, supra). CSP is recombinant full length P.falciparum circumsporozoite protein; AMA-1 is recombinant ectodomain orP. falciparum AMA-1; EBA-175 is recombinant P. falciparum EBA-175rII.

FIG. 8. IFA of infected liver cells in tissue culture. Arrow points toschizont developing in the cell.

FIG. 9. Antibodies from malaria infected people recongnizeLSA-NRC(H)Mut.

DETAILED DESCRIPTION

In the description that follows, a number of terms used in recombinantDNA, parasitology and immunology are extensively utilized. In order toprovide a clearer and consistent understanding of the specification andclaims, including the scope to be given such terms, the followingdefinitions are provided.

In general, an ‘epitope’ is defined as a linear array of 3-20 aminoacids aligned along the surface of a protein. In a linear epitope, theamino acids are joined sequentially and follow the primary structure ofthe protein. In a conformational epitope, residues are not joinedsequentially, but lie linearly along the surface due to the conformation(folding) of the protein. With respect to conformational epitopes, thelength of the epitope-defining sequence can be subject to widevariations. The portions of the primary structure of the antigen betweenthe residues defining the epitope may not be critical to the structureof the conformational epitope. For example, deletion or substitution ofthese intervening sequences may not affect the conformational epitopeprovided sequences critical to epitope conformation are maintained (e.g.cysteines involved in disulfide bonding, glycosylation sites, etc.). Aconformational epitope may also be formed by 2 or more essential regionsof subunits of a homo-oligomer or hetero-oligomer. As used herein,‘epitope’ or ‘antigenic determinant’ means an amino acid sequence thatis immunoreactive. As used herein, an epitope of a designatedpolypeptide denotes epitopes with the same amino acid sequence as theepitope in the designated polypeptide, and immunologic equivalentsthereof. Such equivalents also include strain, subtype (=genotype), ortype(group)-specific variants, e.g. of the currently known sequences orstrains belonging to Plasmodium such as 3D7, FVO, Camp, NF54, and T9/96,or any other known or newly defined Plasmodium strain.

The term ‘solid phase’ intends a solid body to which the individual P.falciparum antigen is bound covalently or by noncovalent means such ashydrophobic, ionic, or van der Waals association.

The term ‘biological sample’ intends a fluid or tissue of a mammalianindividual (e.g. an anthropoid, a human), reptilian, avian, or any otherzoo or farm animal that commonly contains antibodies produced by theindividual, more particularly antibodies against malaria. The fluid ortissue may also contain P. falciparum antigen. Such components are knownin the art and include, without limitation, blood, plasma, serum, urine,spinal fluid, lymph fluid, secretions of the respiratory, intestinal orgenitourinary tracts, tears, saliva, milk, white blood cells andmyelomas. Body components include biological liquids. The term‘biological fluid’ refers to a fluid obtained from an organism.

The term ‘immunologically reactive’ means that the antigen in questionwill react specifically with anti-LSA-1 antibodies, present in vitro orin a body component from a malaria infected individual.

The term ‘immune complex’ intends the combination formed when anantibody binds to an epitope on an antigen.

The term ‘LSA-1’ as used herein refers to the native protein of P.falciparum that contains all the repeats. By LSA-1 is intended LSA-1from any strain of P. falciparum, e.g. 3D7, FVO, Camp, NF54, T9/96, orany other stain.

The term LSA-NRC means the recombinant protein or polypeptide product ofthe gene lsa-nrc that contains a series of amino acids from the P.falciparum native LSA-1 and that comprises an amino acid sequencedefining at least one LSA-1 epitope. We have found that it is necessaryto alter the nucleotide sequence of lsa-nrc such that codon frequency isharmonized for expression in E. coli. It is understood that thenucleotide sequence of the desired lsa-nrc can be altered such thatcodon frequency is harmonized for expression in any desired host.Alternatively, it may not be necessary to harmonize the codon frequencyif the expressed protein levels are satisfactory for the intendedpurpose of the expressed LSA-NRC.

In one aspect of the invention, the LSA-NRC(H) extends fromapproximately amino acid (aa)28 to aa 154, continues with two 17 aatandem repeats and then continues with aa 1630 to 1909 of thefull-length protein (NCBI Genbank accession # A45592). As discussedpreviously, the frequency of amino acid codons of the LSA-1 sequence inthe lsa-nrc^(h) gene construct have been harmonized with the frequencyof E. coli codons in order to improve expression of the protein in E.coli host. It is envisioned that similar codon harmonization can beachieved for enhanced expression of the desired polypeptide in the hostof choice.

The choice of epitopes to be included in a LSA-NRC polypeptide toprovide immunogenic and nonimmunogenic variations can vary depending onthe intended use of the resulting polypeptide. For example, variationsof this peptide can include peptides with repeats from 0 to 90 whereinthe repeats contain ones of the same basic 17 amino acids following theorder: X ¹ GlnGlnX ² AspX ³ GluGlnX ⁴ ArqX ⁵ AlaX ⁶ GluX ⁷ LeuGln (SEQID NO:5) where x₁ is either Glu or Gly; x₂ is Ser or Arg; x₃ is Asp orSer; x₄ is Glu or Asp; x₅ is Leu or Arg; x₆ is Lys or Asn and x₇ is Lysor Thr or Arg. The repeat unit of 17 amino acids can start at any of theamino acids in the 17 amino acid unit, much like the start of a circlecan have many points. However, because of the alpha-helical, coiled-coilnature of the tertiary structure of the repeats each unit shouldmaintain the basic ordering of the amino acids. For example, the secondrepeat unit in LSA-NRC(H) starts following the first repeat with aa #9(Glu) such that the second repeat unit is in order ofGluArgLeuAlaLysGluLysLeuGlnGluGlnGlnArgAspLeuGluGln (SEQ ID NO:2)therefore the amino acid sequence between the N-terminal and C-terminalLSA-1 fragments: GluGlnGlnSerAspLeuGluGlnGluArgLeuAlaLysGluLysLeuGlnGluArgLeuAlaLysGluLysLeuGlnGluGlnGlnArgAspLeuGluGln (SEQ ID NO:24).

Similarly, LSA-NRC polypeptides can also contain less or more of theN-terminal or C-terminal regions of LSA-1 keeping in mind that theseregions contain T-cell epitopes which aid in mounting an immuneresponse. The defined epitopes in these regions, shown in Table 2, canbe included or excluded, depending on the intended purpose of theresulting expressed peptide or polypeptide. The N- and C-terminalregions can additionally be presented, whether complete or partial, inmultiples of 1 in order to achieve a desired immune response. Inaddition, insertions, deletions or substitutions can be designed toenhance immunogenicity or reduce immunogenicity of one or more epitopes.As is described in the Examples below, we have designed LSA-NRC(H)Mutwherein the T5 epitope is interrupted by an insertion of one amino acidand have found that the resulting polypeptide is immunogenic.

The LSA-NRC polypeptide could optionally contain the signal sequencecontained in the first 28 residues of the native protein, and/or anyother amino acid sequence found in other strains or clones of the P.falciparum lsa-1 gene.

The LSA-NRC as used herein also includes analogs and truncated forms ofLSA-NRC that are immunologically cross-reactive with natural LSA-1.

The term ‘homo-oligomer’ as used herein refers to a complex of LSA-NRCcontaining more than one LSA-NRC monomer, e.g. LSA-NRC/LSA-NRC dimers,trimers or tetramers, or any higher-order homo-oligomers of LSA-NRC areall homo-oligomers within the scope of this definition. The oligomersmay contain one, two, or several different monomers of LSA-NRC obtainedfrom the sequence of one strain of P. falciparum or mixed oligomers fromdifferent strains of Plasmodium falciparum including for example 3D7,NF-54, T9/96, Camp, FVO, and others. Such mixed oligomers are stillhomo-oligomers within the scope of this invention, and may allow moreuniversal diagnosis, prophylaxis or treatment of malaria. Thehomo-oligomers may be linked in tandem, head to tail, in random or fixedorder by their ends or cross linked by chemical means by their internalamino acids. Particularly they may be cross-linked by their glutamine(Gln) amino acids by the action of transglutaminase.

The term ‘purified’ as applied to proteins herein refers to acomposition wherein the desired polypeptide comprises at least 35% ofthe total protein component in the composition. The desired polypeptidepreferably comprises at least 40%, more preferably at least about 50%,more preferably at least about 60%, still more preferably at least about70%, even more preferably at least about 80%, even more preferably atleast about 90%, and most preferably at least about 95% of the totalprotein component. The composition may contain other compounds such ascarbohydrates, salts, lipids, solvents, and the like, without affectingthe determination of the percentage purity as used herein. An ‘isolated’LSA-NRC protein intends a Plasmodium protein composition that is atleast 35% pure.

The term ‘essentially purified proteins or polypeptides’ refers toproteins purified such that they can be used for in vitro diagnosticmethods and as a prophylactic compound. These proteins are substantiallyfree from cellular proteins, vector-derived proteins or other Plasmodiumcomponents. The proteins of the present invention are purified tohomogeneity, at least 80% pure, preferably, 90%, more preferably 95%,more preferably 97%, more preferably 98%, more preferably 99%, even morepreferably 99.5%.

The term ‘recombinantly expressed’ used within the context of thepresent invention refers to the fact that the proteins of the presentinvention are produced by recombinant expression methods be it inprokaryotes, or lower or higher eukaryotes as discussed in detail below.

The term ‘lower eukaryote’ refers to host cells such as yeast, fungi andthe like. Lower eukaryotes are generally (but not necessarily)unicellular. Preferred lower eukaryotes are yeasts, particularly specieswithin Saccharomyces. Schizosaccharomyces, Kluveromyces, Pichia (e.g.Pichia pastoris), Hansenula (e.g. Hansenula polymorpha, Yarowia,Schwaniomyces, Schizosaccharomyces, Zygosaccharomyces and the like.Saccharomyces cerevisiae, S. carlsberoensis and K. lactis are the mostcommonly used yeast hosts, and are convenient fungal hosts.

The term ‘prokaryotes’ refers to hosts such as E. coli, Lactobacillus,Lactococcus, Salmonella, Streptococcus, Bacillus subtilis orStreptomyces. Also these hosts are contemplated within the presentinvention.

The term ‘higher eukaryote’ refers to host cells derived from higheranimals, such as mammals, reptiles, insects, and the like. Presentlypreferred higher eukaryote host cells are derived from Chinese hamster(e.g. CHO), monkey (e.g. COS and Vero cells), baby hamster kidney (BHK),pig kidney (PK15), rabbit kidney 13 cells (RK13), the human osteosarcomacell line 143 B, the human cell line HeLa and human hepatoma cell lineslike Hep G2, and insect cell lines (e.g. Spodoptera frugiperda). Thehost cells may be provided in suspension or flask cultures, tissuecultures, organ cultures and the like. Alternatively the host cells mayalso be transgenic animals.

The term ‘polypeptide’ refers to a polymer of amino acids and does notrefer to a specific length of the product; thus, peptides,oligopeptides, and proteins are included within the definition ofpolypeptide. This term also does not refer to or exclude post-expressionmodifications of the polypeptide, for example, glycosylations,acetylations, phosphorylations and the like Included within thedefinition are, for example, polypeptides containing one or moreanalogues of an amino acid (including, for example, unnatural aminoacids, PNA, etc.), polypeptides with substituted linkages, as well asother modifications known in the art, both naturally occurring andnon-naturally occurring.

The term ‘recombinant polynucleotide or nucleic acid’ intends apolynucleotide or nucleic acid of genomic, cDNA, semisynthetic, orsynthetic origin which, by virtue of its origin or manipulation: (1) isnot associated with all or a portion of a polynucleotide with which itis associated in nature, (2) is linked to a polynucleotide other thanthat to which it is linked in nature, or (3) does not occur in nature.

The term ‘recombinant host cells’, ‘host cells’, ‘cells’, ‘cell lines’,‘cell cultures’, and other such terms denoting microorganisms or highereukaryotic cell lines cultured as unicellular entities refer to cellswhich can be or have been, used as recipients for a recombinant vectoror other transfer polynucleotide, and include the progeny of theoriginal cell which has been transfected. It is understood that theprogeny of a single parental cell may not necessarily be completelyidentical in morphology or in genomic or total DNA complement as theoriginal parent, due to natural, accidental, or deliberate mutation.

The term ‘replicon’ is any genetic element, e.g., a plasmid, achromosome, a virus, a cosmid, etc., that behaves as an autonomous unitof polynucleotide replication within a cell; i.e., capable ofreplication under its own control.

The term ‘vector’ is a replicon further comprising sequences providingreplication and/or expression of a desired open reading frame.

The term ‘control sequence’ refers to polynucleotide sequences which arenecessary to effect the expression of coding sequences to which they areligated. The nature of such control sequences differs depending upon thehost organism; in prokaryotes, such control sequences generally includepromoter, ribosomal binding site, and terminators; in eukaryotes,generally, such control sequences include promoters, terminators and, insome instances, enhancers. The term ‘control sequences’ is intended toinclude, at a minimum, all components whose presence is necessary forexpression, and may also include additional components whose presence isadvantageous, for example, leader sequences which govern secretion.

The term ‘promoter’ is a nucleotide sequence that is comprised ofconsensus sequences that allow the binding of RNA polymerase to the DNAtemplate in a manner such that mRNA production initiates at the normaltranscription initiation site for the adjacent structural gene.

The expression ‘operably linked’ refers to a juxtaposition wherein thecomponents so described are in a relationship permitting them tofunction in their intended manner. A control sequence ‘operably linked’to a coding sequence is ligated in such a way that expression of thecoding sequence is achieved under conditions compatible with the controlsequences.

An ‘open reading frame’ (ORF) is a region of a polynucleotide sequencewhich encodes a polypeptide and does not contain stop codons; thisregion may represent a portion of a coding sequence or a total codingsequence.

A ‘coding sequence’ is a polynucleotide sequence that is transcribedinto mRNA and/or translated into a polypeptide when placed under thecontrol of appropriate regulatory sequences. The boundaries of thecoding sequence are determined by a translation start codon at the5′-terminus and a translation stop codon at the 3′-terminus. A codingsequence can include but is not limited to mRNA, DNA (including cDNA),and recombinant polynucleotide sequences.

The term ‘immunogenic’ refers to the ability of a substance to cause ahumoral and/or cellular response, whether alone or when linked to acarrier, in the presence or absence of an adjuvant. ‘Neutralization’refers to an immune response that blocks the infectivity, eitherpartially or fully, of an infectious agent. A ‘vaccine’ is animmunogenic composition capable of eliciting protection against malaria,whether partial or complete. A vaccine may also be useful for treatmentof an infected individual, in which case it is called a therapeuticvaccine.

The term ‘therapeutic’ refers to a composition capable of treatingmalaria infection such that disease symptoms are reduced.

The term ‘effective amount’ for a therapeutic or prophylactic treatmentrefers to an amount of epitope-bearing polypeptide sufficient to inducean immunogenic response in the individual to which it is administered,or to otherwise detectably immunoreact in its intended system (e.g.,immunoassay). Preferably, the effective amount is sufficient to effecttreatment, as defined above. The exact amount necessary will varyaccording to the application. For vaccine applications or for thegeneration of polyclonal antiserum/antibodies, for example, theeffective amount may vary depending on the species, age, and generalcondition of the individual, the severity of the condition beingtreated, the particular polypeptide selected and its mode ofadministration, etc. It is also believed that effective amounts will befound within a relatively large, non-critical range. An appropriateeffective amount can be readily determined using only routineexperimentation. Preferred ranges of LSA-NRC for prophylaxis of malariadisease are about 0.01 to 1000 ug/dose, more preferably about 0.1 to 100ug/dose most preferably about 10-50 ug/dose. Several doses may be neededper individual in order to achieve a sufficient immune response andsubsequent protection against malaria.

More particularly, the present invention contemplates essentiallypurified LSA-NRC and a method for isolating or purifying recombinantLSA-NRC protein.

The term ‘LSA-NRC’ refers to a polypeptide or an analogue thereof (e.g.mimotopes) comprising an amino acid sequence (and/or amino acidanalogues) defining at least one LSA-1 epitope. Typically, the sequencesdefining the epitope correspond to the amino acid sequence of LSA-1region of P. falciparum (either identically, by harmonization, or viasubstitution of analogues of the native amino acid residue that do notdestroy the epitope). The LSA-NRC(H) protein or polypeptide correspondsto a nucleotide sequence identified in SEQ ID NO:22 and an amino acidsequence identified in SEQ ID NO:23 which spans from amino acid 28-154of the N-terminal region, two 17 aa repeats of the 86 possible from thefull length molecule, and the C-terminal region amino acids #1630-1909of LSA-1 3D7 allele. Upon expression in E. coli LSA-NRC(H) is expectedto have an approximate molecular weight of 53 kDa as determined bySDS-PAGE.

The LSA-NRC antigen used in the present invention preferably contains afragment containing at least one complete epitope, or a substantiallyfull-length version, i.e. containing functional fragments thereof (e.g.fragments which are not missing sequence essential to the formation orretention of an epitope). Furthermore, the polypeptide antigen of thepresent invention can also include other sequences that do not block orprevent the formation of the epitope of interest. The presence orabsence of a epitope can be readily determined through screening theantigen of interest with an antibody as described in the Examples below(polyclonal serum or monoclonal to the conformational epitope).

The LSA-NRC polypeptide antigen of the present invention can be made byany recombinant method that provides the epitope of interest. Forexample, recombinant expression in E. coli is a preferred method toprovide non-glycosylated antigens in ‘native’ conformation. This is mostdesirable because natural P. falciparum antigens are not glycosylated.Proteins secreted from mammalian cells may contain modificationsincluding galactose or sialic acids which may be undesirable for certaindiagnostic or vaccine applications. However, it may also be possible andsufficient for certain applications, as it is known for proteins, toexpress the antigen in other recombinant hosts such as baculovirus andyeast or higher eukaryotes, as long as glycosylation is inhibited.

The polypeptides according to the present invention may be secreted orexpressed within compartments of the cell. Preferably, however, thepolypeptides of the present invention are expressed within the cell andare released upon lysing the cells.

It is also understood that the isolates used in the examples section ofthe present invention were not intended to limit the scope of theinvention and that an equivalent sequence from a P. falciparum isolatefrom another allele, e.g. FVO, T9/96 or CAMP, can be used to produce arecombinant LSA-1 protein using the methods described in the presentapplication. Other new strains or clones of P. falciparum may be asuitable source of LSA-1 sequence for the practice of the presentinvention.

The LSA-NRC nucleotide sequence of the present invention can be part ofa recombinant vector. Therefore, the present invention relates moreparticularly to the lsa-nrc^(hmut) nucleic acid sequence (SEQ ID NO:3)in recombinant vector, pET KLSA-NRC^(hmut) deposited with ATCC under theBudapest Treaty on ______, and having accession number ______. The LSA-1genomic sequence was cloned into the base vector pETK(−) a modifiedpET32 plasmid vector from Novagen (Madison, Wis.). This plasmidcomprises, in sequence, a T7 promoter, optionally a lac operator, aribosome binding site, restriction sites to allow insertion of thestructural gene and a T7 terminator sequence. To aid in purification ofthe expressed protein, a single histidine tag is cloned at theC-terminus. The ampicillin antibiotic resistance gene has been replacedwith a kanamycin resistance gene in pETK(−) and the orientation of thekanamycin ORF is opposite to that of the ORF for the gene that isinserted for expression. Examples of other plasmids which contain the T7inducible promoter include the expression plasmids pET-17b, pET-11a,pET-24a-d(+), and pEt-9a, all from Novagen (Madison, Wis.); see theNovagen catalogue.

The present invention also contemplates host cells transformed with arecombinant vector as defined above. In a preferred embodiment, E. colistrain Tuner(DE3), or alternatively BL21 (DE3) (F-ompT hsdSB(rB-mB-) galdcm (DE3) is employed. The above plasmids may be transformed into thisstrain or other strains of E. coli having the following characteristics:a T7 RNA polymerase rec gene, lon, ompT protease mutants or any other E.coli with a protease deficiency such as E. coli origami. Preferably, thehost includes Tuner(DE3) and any of its precursors. Other host cellssuch as insect cells can be used taking into account that other cellsmay result in lower levels of expression.

Eukaryotic hosts include lower and higher eukaryotic hosts as describedin the definitions section. Lower eukaryotic hosts include yeast cellswell known in the art. Higher eukaryotic hosts mainly include mammaliancell lines known in the art and include many immortalized cell linesavailable from the ATCC, including HeLa cells, Chinese hamster ovary(CHO) cells, Baby hamster kidney (BHK) cells, PK15, RK13 and a number ofother cell lines. LSA-NRC expressed in these cells will be glycosylatedunless the cells have been altered such that glycosylation of therecombinant protein is not possible. It is expected that when producingLSA-NRC in a eukaryotic expression system, extensive investigation intomethods for expressing, isolating, purifying, and characterizing theprotein would be required as eukaryotic cells post-translationallymodify this protein and this would alter protein structure andimmunogenicity.

Methods for introducing vectors into cells are known in the art. Pleasesee e.g., Maniatis, Fitsch and Sambrook, Molecular Cloning; A LaboratoryManual (1982) or DNA Cloning, Volumes I and II (D. N. Glover ed. 1985)for general cloning methods.

A preferred method for isolating or purifying LSA-NRC as defined aboveis further characterized as comprising at least the following steps:

(i) growing a host cell as defined above transformed with a recombinantvector expressing LSA-NRC proteins in a suitable culture medium,

(ii) causing expression of said vector sequence as defined above undersuitable conditions for production of a soluble polypeptide,

(iii) lysing said transformed host cells and recovering said LSA-NRCpolypeptide such that it retains its native conformation and isessentially pure.

Once the host has been transformed with the vector, the transformedcells are grown in culture in the presence of the desired antibiotic.For FDA regulatory purposes, it is preferable to use tetracycline orkanamycin. When cells reach optimal biomass density, in this case about0.7-0.9 OD 600, in small culture flasks or 7-100D 600 in bulkfermentors, the cells are induced to produce the recombinant protein,preferably, with dioxin free IPTG. IPTG allows expression of T7polymerase protein from the E. coli (DE3) host cell chromosome, whichacts on the T7 promoter on the expression plasmid. The concentration ofinducer, i.e. IPTG, added affects the maximal protein synthesis. It wasfound that a concentration of 0.5 mM IPTG was best, however, a range of0.25 to 1.0 mM would be sufficient to produce 80-100% of maximal.

The cells are then collected and lysed to release the recombinantpolypeptide. Preferably, lysis is with a microfluidizer, however aFrench Press or other mechanical breakage devise would work;alternatively cells could be lysed by chemical means using urea,guanidine-HCL or detergents such as sarkosyl. Lysed cells arecentrifuged to pellet cellular debris, for example at 10,000 rpm for 30min, and the supernatant is removed and incubated with low levels, 0.1%to 5%, of sarkosyl, a detergent, to facilitate the removal of endotoxin.Lysis is preferably at a temperature of about 0° C.-15° C., morepreferably about 5-10° C. A high salt concentration of about 1.0-2.0 Mis preferable. Salts used include NaCl or other monovalent ions.

Preferably, the E. coli endotoxin is separated and removed from therecombinant polypeptide. This can be done several ways. ForLSA-NRC(H)Mut, a large majority of the endotoxin was removed by applyingthe bacterial lysate directly to a Ni⁺²-NTA column, which bound theLSA-NRC(H)Mut protein. Endotoxin, and other E. coli proteins,carbohydrates and lipopolysaccarides were washed off the column bywashing the resin with a buffer of low pH, about 5.7 to 6.1, preferablyabout pH 5.9 and containing high salt, preferably about 2 M NaCl. Thecell paste to resin ratio can be about 1:8 to about 1:12 w/v, preferablyabout 1:10 w/v. The recombinant protein can be eluted by addition of ahigher pH buffer of about 6.5 to about 7.5, preferably about pH 7.0, ina phosphate buffer of about 10-30 mM, more preferably about 20 mM sodiumphosphate buffer and 300 mM-500 mM imidazole, preferably 400 mM.

At this point the recombinant protein is about 80% pure. If furtherpurity is required, ion-exchange chromatography can be implemented e.g.DEAE and SP-Sepharose. The column is preferably with an ionic charactersuch that the pH used will not promote endotoxin and nucleic acidbinding at the concentration of salt, about 260-300 mM, preferably 280mM, utilized to elute the protein. LSA-NRC(H)Mut was subjected to DEAESepharose ion exchange chromatography. The pH of the buffer can be about6.5 to about 7.5, preferably about 7.0.

Finally, the eluted sample (about 0.1 mg/ml, can be subjected to furtherion exchange chromatography for further concentration and purification.The LSA-NRC(H)Mut was subjected to SP-Sepharose ion exchangechromatography. The pH of the buffer can be about 6.5 to about 7.5,preferably about 7.0. The salt concentration is about 130 mM to about170 mM, preferably about 150 mM.

The present invention also relates to a composition comprising peptidesor polypeptides as described above, for in vitro detection of malariaantibodies present in a biological sample. Therefore, the presentinvention also relates to an LSA-1 specific antibody raised uponimmunizing an animal with a LSA-NRC peptide or protein composition, withsaid antibody being specifically reactive with LSA-1 or LSA-NRC or anyof the polypeptides or peptides as defined above, and with said antibodybeing preferably a monoclonal antibody.

The present invention also relates to an LSA-NRC specific antibodyscreened from a variable chain library in plasmids or phages or from apopulation of human B-cells by means of a process known in the art, withsaid antibody being reactive with any of the polypeptides or peptides asdefined above, and with said antibody being preferably a monoclonalantibody.

The LSA-NRC specific monoclonal antibodies of the invention can beproduced by any hybridoma liable to be formed according to classicalmethods from splenic or lymph node cells of an animal, particularly froma mouse or rat, immunized against the Plasmodium polypeptides orpeptides according to the invention, as defined above on the one hand,and of cells of a myeloma cell line on the other hand, and to beselected by the ability of the hybridoma to produce the monoclonalantibodies recognizing the polypeptides which has been initially usedfor the immunization of the animals.

The antibodies involved in the invention can be labeled by anappropriate label of the enzymatic, fluorescent, or radioactive type.

The monoclonal antibodies according to this preferred embodiment of theinvention may be humanized versions of mouse monoclonal antibodies madeby means of recombinant DNA technology, departing from parts of mouseand/or human genomic DNA sequences coding for H and L chains from cDNAor genomic clones coding for H and L chains.

Alternatively the monoclonal antibodies according to this preferredembodiment of the invention may be human monoclonal antibodies. Theseantibodies according to the present embodiment of the invention can alsobe derived from human peripheral blood lymphocytes of patients infectedwith malaria, or vaccinated against malaria. Such human monoclonalantibodies are prepared, for instance, by means of human peripheralblood lymphocytes (PBL) repopulation of severe combined immunedeficiency (SCID) mice, or by means of transgenic mice in which humanimmunoglobulin genes have been used to replace the mouse genes.

The invention also relates to the use of the proteins or peptides of theinvention, for the selection of recombinant antibodies by the process ofrepertoire cloning.

Antibodies directed to peptides or single or specific proteins derivedfrom a certain strain may be used as a medicament, more particularly forincorporation into an immunoassay for the detection of Plasmodiumstrains for detecting the presence of LSA-1 antigens, or antigenscontaining LSA-NRC epitopes, for prognosing/monitoring of malariadisease, or as therapeutic agents.

Alternatively, the present invention also relates to the use of any ofthe above-specified LSA-NRC monoclonal antibodies for the preparation ofan immunoassay kit for detecting the presence of LSA-1 antigen orLSA-NRC antigens containing LSA-1 epitopes in a biological samples forthe preparation of a kit for prognosing/monitoring of malaria disease orfor the preparation of a malaria medicament.

The present invention also relates to a method for in vitro diagnosis ordetection of malaria antigen present in a biological sample, comprisingat least the following steps:

(i) contacting said biological sample with any of the LSA-NRC specificmonoclonal antibodies as defined above, preferably in an immobilizedform under appropriate conditions which allow the formation of an immunecomplex,

(ii) removing unbound components,

(iii) incubating the immune complexes formed with heterologousantibodies, which specifically bind to the antibodies present in thesample to be analyzed, with said heterologous antibodies conjugated to adetectable label under appropriate conditions,

(iv) detecting the presence of said immune complexes visually ormechanically (e.g. by means of densitometry, fluorimetry, colorimetry).

The present invention also relates to a kit for in vitro diagnosis of amalaria antigen present in a biological sample, comprising:

at least one monoclonal antibody as defined above, with said antibodybeing preferentially immobilized on a solid substrate,

a buffer or components necessary for producing the buffer enablingbinding reaction between these antibodies and the malaria antigenspresent in the biological sample, and

a means for detecting the immune complexes formed in the precedingbinding reaction.

The kit can possibly also include an automated scanning andinterpretation device for inferring the malaria antigens present in thesample from the observed binding pattern.

Monoclonal antibodies according to the present invention are suitableboth as therapeutic and prophylactic agents for treating or preventingmalaria infection in susceptible malaria-infected subjects.

In general, this will comprise administering a therapeutically orprophylactically effective amount of one or more monoclonal antibodiesof the present invention to a susceptible subject or one exhibitingmalaria infection. Any active form of the antibody can be administered,including Fab and F(ab′)₂ fragments. Antibodies of the present inventioncan be produced in any system, including insect cells, baculovirusexpression systems, chickens, rabbits, goats, cows, or plants such astomato, potato, banana or strawberry. Methods for the production ofantibodies in these systems are known to a person with ordinary skill inthe art. Preferably, the antibodies used are compatible with therecipient species such that the immune response to the MAbs does notresult in clearance of the MAbs before parasite can be controlled, andthe induced immune response to the MAbs in the subject does not induce“serum sickness” in the subject. Preferably, the MAbs administeredexhibit some secondary functions such as binding to Fc receptors of thesubject.

Treatment of individuals having malaria infection may comprise theadministration of a therapeutically effective amount of LSA-NRCantibodies of the present invention. The antibodies can be provided in akit as described below. The antibodies can be used or administered as amixture, for example in equal amounts, or individually, provided insequence, or administered all at once. In providing a patient withantibodies, or fragments thereof, capable of binding to LSA-1, or anantibody capable of protecting against malaria in a recipient patient,the dosage of administered agent will vary depending upon such factorsas the patient's age, weight, height, sex, general medical condition,previous medical history, etc.

In general, it is desirable to provide the recipient with a dosage ofantibody which is in the range of from about 1 pg/kg-100 pg/kg, 100pg/kg-500 pg/kg, 500 pg/kg-1 ng/kg, 1 ng/kg-100 ng/kg, 100 ng/kg-500ng/kg, 500 ng/kg-1 ug/kg, 1 ug/kg-100 ug/kg, 100 ug/kg-500 ug/kg, 500ug/kg-1 mg/kg, 1 mg/kg-50 mg/kg, 50 mg/kg-100 mg/kg, 100 mg/kg-500mg/kg, 500 mg/kg-1 g/kg, 1 g/kg-5 g/kg, 5 g/kg-10 g/kg (body weight ofrecipient), although a lower or higher dosage may be administered.

In a similar approach, another prophylactic use of the monoclonalantibodies of the present invention is the active immunization of apatient using an anti-idiotypic antibody raised against one of thepresent monoclonal antibodies. Immunization with an anti-idiotype whichmimics the structure of the epitope could elicit an active anti-LSA-NRCresponse (Linthicum, D. S. and Farid, N. R., Anti-Idiotypes, Receptors,and Molecular Mimicry (1988), pp 1-5 and 285-300).

Likewise, active immunization can be induced by administering one ormore antigenic and/or immunogenic epitopes as a component of a subunitvaccine. Vaccination could be performed orally or parenterally inamounts sufficient to enable the recipient to generate protectiveantibodies or immunoreactive T-cells against LSA-1 in a manner that haseither prophylactical or therapeutical value. The host can be activelyimmunized with the antigenic/immunogenic peptide in pure form, afragment of the peptide, or a modified form of the peptide. One or moreamino acids, not corresponding to the original protein sequence can beadded to the amino or carboxyl terminus of the original peptide, ortruncated form of peptide. Such extra amino acids are useful forcoupling the peptide to another peptide, to a large carrier protein, orto a support. Amino acids that are useful for these purposes include:tyrosine, lysine, glutamic acid, aspartic acid, cyteine and derivativesthereof. Alternative protein modification techniques may be used e.g.,NH₂-acetylation or COOH-terminal amidation, to provide additional meansfor coupling or fusing the peptide to another protein or peptidemolecule or to a support.

The antibodies capable of protecting against malaria are intended to beprovided to recipient subjects in an amount sufficient to effect areduction in the malaria infection symptoms. An amount is said to besufficient to “effect” the reduction of infection symptoms if thedosage, route of administration, etc. of the agent are sufficient toinfluence such a response. Responses to antibody administration can bemeasured by analysis of subject's vital signs.

The present invention more particularly relates to a compositioncomprising at least one of the above-specified peptides or a recombinantLSA-NRC protein or polypeptide composition as defined above, for use asa vaccine for immunizing a mammal, preferably humans, against malaria,comprising administering a sufficient amount of the composition possiblyaccompanied by pharmaceutically acceptable adjuvant(s), to produce animmune response.

The proteins of the present invention, preferably purified recombinantLSA-NRC from one or more alleles of P. falciparum, e.g., T9/96, FVO,3D7, NF54, or CAMP, are expected to provide a particularly usefulvaccine antigen, since the antigen has very little sequence variation inthe N-terminal and C-terminal regions that contain the T-cell epitopes.

Pharmaceutically acceptable carriers include any carrier that does notitself induce the production of antibodies harmful to the individualreceiving the composition. Suitable carriers are typically large, slowlymetabolized macromolecules such as proteins, polysaccharides, polylacticacids, polyglycolic acids, polymeric amino acids, amino acid copolymers;and inactive virus particles. Such carriers are well known to those ofordinary skill in the art.

Preferred adjuvants to enhance effectiveness of the composition include,but are not limited to: T helper cell type I adjuvant, Montanide,aluminum hydroxide (alum), N-acetyl-muramyl-L-threonyl-D-isoglutamine(thr-MDP) as found in U.S. Pat. No. No. 4,606,918,N-acetyl-normuramyl-L-alanyl-D-isoglutamine(nor-MDP),N-acetylmuramyl-L-alanyl-D-isoglutaminyl-L-alanine-2-(1′-2′-dipalmitoyl-sn-glycero-3-hydroxyphosphoryloxy)-ethylamine(MTP-PE) and RIBI, which contains three components extracted frombacteria, monophosphoryl lipid A, trehalose dimycolate, and cell wallskeleton (MPL+TDM+CWS) in a 2% squalene/Tween 80 emulsion. Any of the 3components MPL, TDM or CWS may also be used alone or combined 2 by 2.Additionally, adjuvants such as Stimulon (Cambridge Bioscience,Worcester, Mass.) or SAF-1 (Syntex) may be used. Further, CompleteFreund's Adjuvant (CFA) and Incomplete Freund's Adjuvant (IFA) may beused for non-human applications and research purposes.

All documents cited herein supra and infra are hereby incorporated byreference thereto.

The immunogenic compositions typically will contain pharmaceuticallyacceptable vehicles, such as water, saline, glycerol, ethanol, etc.Additionally, auxiliary substances, such as wetting or emulsifyingagents, pH buffering substances, preservatives, and the like, may beincluded in such vehicles.

Typically, the immunogenic compositions are prepared as injectables,either as liquid solutions or suspensions; solid forms suitable forsolution in, or suspension in, liquid vehicles prior to injection mayalso be prepared. The preparation also may be emulsified or encapsulatedin liposomes for enhanced adjuvant effect. The LSA-NRC proteins of theinvention may also be incorporated into Immune Stimulating Complexestogether with saponins, for example QuilA (ISCOMS).

Immunogenic compositions used as vaccines comprise a ‘sufficient amount’or ‘an immunologically effective amount’ of the proteins of the presentinvention, as well as any other of the above mentioned components, asneeded. ‘Immunologically effective amount’, means that theadministration of that amount to an individual, either in a single doseor as part of a series, is effective for treatment, as defined above.This amount varies depending upon the health and physical condition ofthe individual to be treated, the taxonomic group of individual to betreated (e.g. nonhuman primate, primate, etc.), the capacity of theindividual's immune system to synthesize antibodies, the degree ofprotection desired, the formulation of the vaccine, the treatingdoctor's assessment of the medical situation, the strain of malariainfection, and other relevant factors. It is expected that the amountwill fall in a relatively broad range that can be determined throughroutine trials. Usually, the amount will vary from 0.01 to 1000 ug/dose,more particularly from about 1.0 to 100 ug/dose most preferably fromabout 10 to 50 ug/dose.

The proteins may also serve as vaccine carriers to present homologous(e.g. other malaria antigens, such as EBA-175 or AMA-1) or heterologous(non-malaria) antigens. In this use, the proteins of the inventionprovide an immunogenic carrier capable of stimulating an immune responseto other antigens. For example, the polypeptide can be included in acomposition containing other P. falciparum malaria proteins includingRTS,S (Stoute et al., 1998. J. Infect. Dis. 178(4):1139-44), AMA-1(Dutta et al., 2002 Infect Immun. 70(6): 3101-10), MSP-1 (Angov, et al.2003. Mol Biochem Parasitol. 128:195-204), or P. vivax malaria proteinslike MSP-1 (Dutta, et al. 2001. Infect Immun 69, 5464-5470) or DBP(Dutta et al, 2000. Mol BioChem Para. 109/2;179-85). The P. falciparumLSA-NRC polypeptide could also provide immune help or boosting to othervaccine constructs that might contain P. falciparum LSA-NRC epitopes.These other vaccine constructs could be of protein (e.g. a synthetic orrecombinant amino acid construct containing LSA-1 sequences), nucleicacid (e.g. naked DNA plasmid vector containing LSA-1 gene fragments) orlive viral (e.g. poxvirus, adenovirus or VEE virus geneticallyengineered to contain LSA-1 DNA sequences) or bacterial (e.g. Salmonellaor Mycobacterium cells containing extra chromosomal plasmids orchromosomally integrated DNA sequences of LSA-1) vectors in origin suchthat they are delivered to the intended host as a DNA sequence thatwould be translated into a polypeptide that contain sequences similarto, or contained in, the LSA-NRC polypeptide. The P. falciparum LSA-NRCpolypeptide could also provide immune help in a vaccine by being in thefirst, second or third priming injections that are followed by anothervaccine construct (booster injection) that would contain P. falciparumLSA-NRC epitopes. The booster injection could also be the P. falciparumLSA-NRC polypeptide in a different adjuvant than it was emulsified in orcombined with for the priming injection or injections.

The antigen may be conjugated either by conventional chemical methods,or may be cloned into the gene encoding LSA-NRC fused to the 5′ end orthe 3′ end of the LSA-NRC gene. The vaccine may be administered inconjunction with other immunoregulatory agents.

The compounds of the present invention can be formulated according toknown methods to prepare pharmaceutically useful compositions, wherebythese materials, or their functional derivatives, are combined inadmixture with a pharmaceutically acceptable carrier vehicle. Suitablevehicles and their formulation, inclusive of other human proteins, e.g.,human serum albumin, are described, for example, in Remington'sPharmaceutical Sciences (16th ed., Osol, A. ed., Mack Easton Pa.(1980)). In order to form a pharmaceutically acceptable compositionsuitable for effective administration, such compositions will contain aneffective amount of the above-described compounds together with asuitable amount of carrier vehicle.

Additional pharmaceutical methods may be employed to control theduration of action. Control release preparations may be achieved throughthe use of polymers to complex or absorb the compounds. The controlleddelivery may be exercised by selecting appropriate macromolecules (forexample polyesters, polyamino acids, polyvinyl, pyrrolidone,ethylenevinylacetate, methylcellulose, carboxymethylcellulose, orprotamine sulfate) and the concentration of macromolecules as well asthe method of incorporation in order to control release. Anotherpossible method to control the duration of action by controlled releasepreparations is to incorporate the compounds of the present inventioninto particles of a polymeric material such as polyesters, polyaminoacids, hydrogels, poly(lactic acid) or ethylene vinylacetate copolymers.Alternatively, instead of incorporating these agents into polymericparticles, it is possible to entrap these materials in microcapsulesprepared, for example, interfacial polymerization, for example,hydroxymethylcellulose or gelatin-microcapsules andpoly(methylmethacylate)-microcapsules, respectively, or in colloidaldrug delivery systems, for example, liposomes, albumin microspheres,microemulsions, nanoparticles, and nanocapsules or in macroemulsions.Such techniques are disclosed in Remington's Pharmaceutical Sciences(1980).

Administration of the compounds, whether antibodies or vaccines,disclosed herein may be carried out by any suitable means, includingparenteral injection (such as intraperitoneal, subcutaneous, orintramuscular injection), orally, or by topical application of theantibodies (typically carried in a pharmaceutical formulation) to anairway surface. Topical application of the antibodies to an airwaysurface can be carried out by intranasal administration (e.g., by use ofdropper, swab, or inhaler which deposits a pharmaceutical formulationintranasally). Topical application of the antibodies to an airwaysurface can also be carried out by inhalation administration, such as bycreating respirable particles of a pharmaceutical formulation (includingboth solid particles and liquid particles) containing the antibodies asan aerosol suspension, and then causing the subject to inhale therespirable particles. Methods and apparatus for administering respirableparticles of pharmaceutical formulations are well known, and anyconventional technique can be employed. Oral administration may be inthe form of an ingestable liquid or solid formulation.

The treatment may be given in a single dose schedule, or preferably amultiple dose schedule in which a primary course of treatment may bewith 1-10 separate doses, followed by other doses given at subsequenttime intervals required to maintain and or reinforce the response, forexample, at 1-4 months for a second dose, and if needed, a subsequentdose(s) after several months. Examples of suitable treatment schedulesinclude: (i) 0, 1 month and 6 months, (ii) 0, 7 days and 1 month, (iii)0 and 1 month, (iv) 0 and 6 months, or other schedules sufficient toelicit the desired responses expected to reduce disease symptoms, orreduce severity of disease.

The present invention also provides kits which are useful for carryingout the present invention. The present kits comprise a first containermeans containing the above-described vaccine. The kit also comprisesother container means containing solutions necessary or convenient forcarrying out the invention. The container means can be made of glass,plastic or foil and can be a vial, bottle, pouch, tube, bag, etc. Thekit may also contain written information, such as procedures forcarrying out the present invention or analytical information, such asthe amount of reagent contained in the first container means. Thecontainer means may be in another container means, e.g. a box or a bag,along with the written information.

The present invention also relates to a method for in vitro diagnosis ofmalaria antibodies present in a biological sample, comprising at leastthe following steps

(i) contacting said biological sample with a composition comprising anyof the LSA-NRC recombinant protein or peptides as defined above,preferably in an immobilized form under appropriate conditions whichallow the formation of an immune complex, wherein said peptide orprotein can be a biotinylated peptide or protein which is covalentlybound to a solid substrate by means of streptavidin or avidin complexes,

(ii) removing unbound components,

(iii) incubating the immune complexes formed with heterologousantibodies, with said heterologous antibodies having conjugated to adetectable label under appropriate conditions,

(iv) detecting the presence of said immune complexes visually ormechanically (e.g. by means of densitometry, fluorimetry, colorimetry).

The present invention also relates to a kit for determining the presenceof malaria antibodies, in a biological sample, comprising:

(i) at least one peptide or protein composition as defined above,possibly in combination with other polypeptides or peptides fromPlasmodium or other types of malaria parasite, with said peptides orproteins being preferentially immobilized on a solid support, morepreferably on different microwells of the same ELISA plate, and evenmore preferentially on one and the same membrane strip,

(ii) a buffer or components necessary for producing the buffer enablingbinding reaction between these polypeptides or peptides and theantibodies against malaria present in the biological sample,

(iii) means for detecting the immune complexes formed in the precedingbinding reaction,

(iv) possibly also including an automated scanning and interpretationdevice for inferring the malaria parasite present in the sample from theobserved binding pattern.

The immunoassay methods according to the present invention utilizeLSA-NRC domains that maintain linear and conformational epitopesrecognized by antibodies in the sera from individuals infected with amalaria parasite. The LSA-NRC antigens of the present invention may beemployed in virtually any assay format that employs a known antigen todetect antibodies. A common feature of all of these assays is that theantigen is contacted with the body component suspected of containingmalaria antibodies under conditions that permit the antigen to bind toany such antibody present in the component. Such conditions willtypically be physiologic temperature, pH and ionic strength using anexcess of antigen. The incubation of the antigen with the specimen isfollowed by detection of immune complexes comprised of the antigen andantibody.

Design of the immunoassays is subject to a great deal of variation, andmany formats are known in the art. Protocols may, for example, use solidsupports, or immunoprecipitation. Most assays involve the use of labeledantibody or polypeptide; the labels may be, for example, enzymatic,fluorescent, chemiluminescent, radioactive, or dye molecules. Assayswhich amplify the signals from the immune complex are also known;examples of which are assays which utilize biotin and avidin orstreptavidin, and enzyme-labeled and mediated immunoassays, such asELISA assays.

The immunoassay may be, without limitation, in a heterogeneous or in ahomogeneous format, and of a standard or competitive type. In aheterogeneous format, the polypeptide is typically bound to a solidmatrix or support to facilitate separation of the sample from thepolypeptide after incubation. Examples of solid supports that can beused are nitrocellulose (e.g., in membrane or microtiter well form),polyvinyl chloride (e.g., in sheets or microtiter wells), polystyrenelatex (e.g., in beads or microtiter plates, polyvinylidine fluoride(known as Immunolon.™.), diazotized paper, nylon membranes, activatedbeads, and Protein A beads. For example, Dynatech Immunolon.™.1 orImmunlon.™. 2 micrometer plates or 0.25 inch polystyrene beads(Precision Plastic Ball) can be used in the heterogeneous format. Thesolid support containing the antigenic polypeptides is typically washedafter separating it from the test sample, and prior to detection ofbound antibodies. Both standard and competitive formats are known in theart.

In a homogeneous format, the test sample is incubated with thecombination of antigens in solution. For example, it may be underconditions that will precipitate any antigen-antibody complexes whichare formed. Both standard and competitive formats for these assays areknown in the art.

In a standard format, the amount of malaria antibodies in theantibody-antigen complexes is directly monitored. This may beaccomplished by determining whether labeled anti-xenogeneic (e.g.anti-human) antibodies which recognize an epitope on anti-malariaantibodies will bind due to complex formation. In a competitive format,the amount of malaria antibodies in the sample is deduced by monitoringthe competitive effect on the binding of a known amount of labeledantibody (or other competing ligand) in the complex.

Complexes formed comprising anti-malaria antibody (or in the case ofcompetitive assays, the amount of competing antibody) are detected byany of a number of known techniques, depending on the format. Forexample, unlabeled malaria antibodies in the complex may be detectedusing a conjugate of anti-xenogeneic Ig complexed with a label (e.g. anenzyme label).

In an immunoprecipitation or agglutination assay format the reactionbetween the malaria antigens and the antibody forms a network thatprecipitates from the solution or suspension and forms a visible layeror film of precipitate. If no anti-malaria antibody is present in thetest specimen, no visible precipitate is formed.

There currently exist three specific types of particle agglutination(PA) assays. These assays are used for the detection of antibodies tovarious antigens when coated to a support. One type of this assay is thehemagglutination assay using red blood cells (RBCs) that are sensitizedby passively adsorbing antigen (or antibody) to the RBC. The addition ofspecific antigen antibodies present in the body component, if any,causes the RBCs coated with the purified antigen to agglutinate.

To eliminate potential non-specific reactions in the hemagglutinationassay, two artificial carriers may be used instead of RBC in the PA. Themost common of these are latex particles. However, gelatin particles mayalso be used. The assays utilizing either of these carriers are based onpassive agglutination of the particles coated with purified antigens.

The LSA-NRC proteins, polypeptides, or antigens of the present inventionwill typically be packaged in the form of a kit for use in theseimmunoassays. The kit will normally contain in separate containers theLSA-NRC antigen, control antibody formulations (positive and/ornegative), labeled antibody when the assay format requires the same andsignal generating reagents (e.g. enzyme substrate) if the label does notgenerate a signal directly. The LSA-NRC antigen may be already bound toa solid matrix or separate with reagents for binding it to the matrix.Instructions (e.g. written, tape, CD-ROM, etc.) for carrying out theassay usually will be included in the kit.

Immunoassays that utilize the LSA-NRC antigen are useful in screeningblood for the preparation of a supply from which potentially infectivemalaria parasite is lacking. The method for the preparation of the bloodsupply comprises the following steps. Reacting a body component,preferably blood or a blood component, from the individual donatingblood with LSA-NRC proteins of the present invention to allow animmunological reaction between malaria antibodies, if any, and theLSA-NRC antigen. Detecting whether anti-malaria antibody—LSA-NRC antigencomplexes are formed as a result of the reacting. Blood contributed tothe blood supply is from donors that do not exhibit antibodies to thenative LSA-NRC antigens.

The contents of all cited references (including literature references,issued patents, published patent applications, and co-pending patentapplications) cited throughout this application are hereby expresslyincorporated by reference.

Other features of the invention will become apparent in the course ofthe following descriptions of exemplary embodiments which are given forillustration of the invention and are not intended to be limitingthereof.

The following Materials and Methods were used in the Examples below.

Cloning and Expression.

Two gene constructs containing modified codons to encode for theN-terminal (#28-154 residues), the C-terminal (#1630-1909 residues) andtwo 17 amino acid repeats of LSA-1 of the P. falciparum 3D7 clone(residue numbers refer to Genbank protein sequence for 3D7 clone, ID #A45592) were commercially synthesized (Retrogen, San Diego, Calif.). Thefirst gene construct (lsa-nrc^(e)) was designed based on the codon usageof the highly expressed genes in E. coli cells and the second geneconstruct (lsa-nrc^(h)) was designed to “harmonize” translation rates,as predicted by comparison of codon frequency tables between P.falciparum and E. coli (Angov et al, U.S. patent application Ser. No.10/440,668 filed on 1 Apr. 2003). We chose one copy of the major 17amino acid repeats, GluGlnGlnSerAspLeuGluGlnGluArgLeuAlaLysGluLysLeuGln,(SEQ ID NO:1) that occurs 31 times in 3D7 clone protein and one copy ofa minor repeat, GluGlnGlnArgAspLeuGluGlnGluArgLeuAlaLysGluLysLeuGln (SEQID NO:2), that occurs 4 times in 3D7 clone protein. The synthetic geneswere ligated into the Nde I, Not I sites of the modified pET32 plasmidpET K—. The resultant plasmid construct was designated pET KLSA-NRC^(e)or pET KLSA-NRC^(h) (later referred to as pET KLSA-NRC^(hmut)) and eachrecombinant plasmid was transformed into E. coli DH5α cells. The insertwas sequenced on both strands of DNA. For protein expression the plasmidwas transformed into an E. coli host strain Tuner (DE3) (Novagen,Madison, Wis.). The transformations were plated onto LB Agar plates with50 pg/ml kanamycin. The pET KLSA-NRC^(e) was unstable in Tuner (DE3)cells as well as Bl21(DE3), HMS174(DE3). Protein expression was low andplasmids could not be maintained more than several passages. However thepET KLSA-NRC^(hmut) plasmid was stable and expression of LSA-NRC(H)Mutpolypeptide was good. The expression of LSA-NRC(H)Mut was confirmed byIPTG induction in shake flask cultures and cell banks were prepared byinoculating LB supplemented with 1% glucose and 35 μg/ml kanamycin.Cultures were grown to an OD 600=1 and cryopreserved in 8% glycerol andwere used to prepare the inocula for bulk fermentation.

Fermentation

The expression of LSA-NRC(H)Mut was performed in a 10-liter bioreactor(New Brunswick Scientific, Edison, N.J.) at the lab scale and in a300-liter bioreactor (New Brunswick Scientific) at the WRAIR Departmentof Biologics Research, Pilot Production Facility. To prepare E. colicell paste containing LSA-NRC(H)Mut Select APS Super broth mediumcontaining 35 μg/ml kanamycin and 1% glucose was inoculated with a freshstationary phase culture in accordance with BPR-670-00. The culture isgrown at 37±1EC to an optical density of 7-80D and induced with 0.5 mMIPTG for 2±0.25 hrs. The cell paste is harvested by centrifugation at15,000 rpm and stored frozen at −80±10° C.

Plasmid Stability

The presence of recombinant plasmid in E. coli Tuner (DE3) cells afterfermentation was determined by plating an appropriate dilution of cellson LB agar plates containing either kanamycin 50 ug/ml (selectiveplates) and on LB agar plates containing no antibiotic (non-selectiveplates). The percent plasmid retention was calculated using colonycounts on appropriate dilution plates containing between 30 and 300colonies.

Metal Affinity Purification

All buffers were maintained at 4±2EC; all chemicals used duringpurification were ACS certified or the next best available grade.Purification was carried out at RT on a Waters-600 liquid chromatographysystem. Cell paste was thawed and suspended in 25 times w/v of Buffer A(50 mM sodium phosphate (NaP), 2 M NaCl, pH 5.9) and mixed untilhomogenous. This suspension was mixed and the E. coli cells weredisrupted by high-pressure microfluidization (Model 1109, MicrofluidicCorp., Newton, Mass.). The cell lysate was cleared by centrifugation at10,000×g and Buffer B (20% sodium N-lauroyl sarcosine) (sarkosyl) wasadded to a final concentration of 0.5% and incubated at 4±2EC for 30 minbefore loading onto a Ni⁺²-NTA Superflow column (Qiagen, Valencia,Calif.; 10 ml packed resin per gram paste). The Ni⁺²-NTA column waspre-equilibrated with buffer-C (Buffer A containing 0.5% sarkosyl; pH5.9). After loading the lysate, the Ni⁺²-NTA resin was washed with 40column volumes (CV) of Buffer D (Buffer A with 5 mM imidazole, 0.5%sarkosyl; pH 5.9) followed by 40 CV of Buffer E (20 mM NaP, 5 mMimidazole, 75 mM NaCl; pH 7.0). Bound proteins were eluted from thecolumn in Buffer F containing 300 mM imidazole (pH 7.0).

Ion-Exchange Purification

Ion-exchange column resins were sanitized with 0.2 N NaOH before use andthen equilibrated to initial binding conditions. The protein wasconcentrated on a DEAE Sepharose anion-exchange column (AmershamPharmacia Biotech, Piscataway, N.J.; 3 ml packed resin per gram ofstarting bacterial paste). The column was pre-equilibrated with Buffer G(Buffer F without imidazole). After loading the protein, the column waswashed with 10 CV of Buffer H (20 mM NaP, 200 mM NaCl, pH 7.0).LSA-NRC(H)Mut was eluted in buffer-I containing a final concentration of280 mM NaCl (pH 7.0). LSA-NRC(H)Mut eluted from the DEAE column wasdiluted to 50 mM NaCl, pH 7.0 and loaded on an SP Sepharosecation-exchange column (Amersham Pharmacia Biotech; 2 ml packed resinper gram paste), pre-equilibrated with buffer-K (20 mM NaP, 50 mM NaCl;pH 7.0). The column was washed with 20 CV of Buffer-K. LSA-NRC(H)Mut waseluted from the column in Buffer L (20 mM NaP, 150 mM NaCl, pH 7.0).

Formulation, Lyophilization and Storage

Purified LSA-NRC(H)Mut protein eluted from the SP column was quantifiedby Bio-Rad protein assay (BioRad, Richmond, Calif.). LSA-NRC(H)Mut wasvialed at 100 μg ml⁻¹, 65 μg protein per vial, in the final formulationbuffer (23.5 mM NaH₂PO₄.H₂O, 30 mM NaCl, 0.1 mM EDTA, 3.15% sucrose; pH7.1) and lyophilized.

Residual Sarkosyl and Endotoxin Content Determination

The residual sarkosyl in purified LSA-NRC(H)Mut protein preparation wasmeasured by a reversed-phase HPLC method. Endotoxin content wasestimated using the chromogenic Limulus Amebocyte Lysate (LAL) kineticassay (Associates of Cape Cod, Falmouth, Mass.). Dilutions of allprotein samples and LAL standard were prepared in pyrogen-free 96 wellplates. Positive control solutions prepared for the standard curvesranged from 1 endotoxin unit (EU) ml⁻¹ to 0.06 EU ml⁻¹, in two-foldserial dilutions. The assay was carried out as per the manufacturer'sinstructions and the 96-well plates were read at 405 nm on V_(max)kinetic microplate reader at 2 min intervals for 60 min (MolecularDevices Corp., Sunnyvale, Calif.).

Purity and Stability Analysis

LSA-NRC(H)Mut was evaluated for purity on precast polyacrylamide gels(4-12% gradient Bis-Tris, Invitrogen, Carlsbad, Calif.), 0.1-16 μgprotein loaded per well. Gels were stained with Coomassie blue orGelCode SilverSNAP stain (Pierce, Rockford, Ill.), destained, scanned ona Laser densitometer and acquired data was analyzed by ImageQuant 5.1software (Molecular Dynamics, Sunnyvale, Calif.). Residual host cellprotein (HCP) content, was assessed by ELISA and Western blotting, usingcommercially available kits (Cygnus Technologies, Plainville, Mass.).The HCP standard recommended by the manufacturer was used. In additionto this control, a lysate of the E. coli host was tested as a standardbetween 1000 and 15 ng ml⁻¹ protein concentration, to determine if thekit was capable of detecting proteins from this specific host E. coli.Immunoblotting for HCP determination (Cygnus Technologies kit) wascarried out using the HCP standard provided by the manufacturer. Theproteins were electrophoretically transferred to a nitrocellulosemembrane and the western blot assay was performed as per themanufacturer's instructions. Stability of LSA-NRC was determined bySDS-PAGE and western blotting of protein samples drawn monthly fromaliquots stored at −80° C., −30° C., 4° C., 22° C. (RT) and 37° C.

Gel-Permeation (GPC) and Reversed-Phase (RPC) Chromatography

HPLC analysis of purified protein was carried out using a Waters-510HPLC pump, connected to Waters-712 WISP autosampler and controlled byMillenium Release 3.2 chromatographic software (Waters Corp., Milford,Mass.). Waters-996 PDA detector was used to monitor the elutionprofiles. For GPC analysis a Shodex Protein KW-803 column (Waters Corp.,Milford, Mass.) was used with 10 μg protein injection. Buffer systemconsisted of 20 mM sodium phosphate, 100 mM K₂SO₄ (pH 7.15) at 0.5 mlmin⁻¹ flow rate. The column was calibrated with molecular weightstandards (BioRad). RPC analysis was done with a C8 Aquapore RP-300 Åcolumn, 7μ, 30×2.1 mm (PE Brownlee, Norwalk, Conn.) at 0.5 ml min⁻¹flow-rate and 4-12 μg protein per load. Solvent A: 0.05% trifluroaceticacid (TFA) in H₂O; solvent B: 0.05% TFA in acetonitrile. The solventgradient consisted of 100% solvent A for 5 min, 100% to 30% solvent Aover 15 min, 30% to 0% solvent A over 5 min and back to 100% solvent Aover 5 min.

LSA-NRC(H)Mut Animal Immunizations:

Groups of five Balb/c, C57BL/6 and C57BL/7-TgN (HLA2.1) female mice(male and females for −TgN mice), 8-10 weeks old, were immunized threetimes with formulations containing 0.1, 1.0 and 10 μg of LSA-NRC(H)Mutand Montanide ISA 720® (Seppic, France) as adjuvant. A group immunizedwith adjuvant alone was used as control. Each formulation was preparedas a 100 μl/dose and given subcutaneously at 0, 4 and 8 wks. Serumsamples were taken two weeks after each immunization and tested forspecific LSA-NRC(H)Mut antibodies. One or two mice were randomlyselected from each group 7 days after the second immunization and theirspleens were removed. Spleen cells harvested, pooled and tested forinterferon gamma (IFN-gamma) production as described below.

Rabbit Immunizations

Three NZW rabbits, 3 months old, were immunized subcutaneously threetimes with 1-ml formulations containing 100 μg LSA-NRC(H)Mut/MontanideISA 720. Immunizations were given at 0, 4 and 8 wks and serum sampleswere obtained two weeks after each immunization.

Human Serum

Human serum samples were obtained from adult individuals living inmalaria endemic areas in Kenya and from healthy volunteers exposed toirradiated P. falciparum sporozoites as part of a vaccine trial (Hoffmanet al, 2002, supra.).

ELISA.

Mouse and rabbit sera were screened for specific LSA-NRC(H)Mut antibodyproduction on ELISA. Briefly, 96-well microtiter plates (Dynax,Chantilly, Va.) were coated with 50 ng per well of either LSA-NRC^(H),incubated overnight at 4° C., blocked for 1 hour with 1×PBS-0.05% Tween20 (PBST) containing 5% casein (Sigma, St. Louis, Mo.), and washed fourtimes with PBST. Plates were incubated for 1 hr at room temperature, RT,with consecutive dilutions of sera starting at 1:50. The plates werewashed four times with PBST and incubated with 1:4000 dilution ofanti-mouse or anti-rabbit IgG HRP-conjugated secondary antibody(Southern biotech) for 1 hr, RT. Then they again were washed four timesand developed for 30 min with ABTS[2,2′-azinobis(3-ethylbenzthiazolinesulfonic acid)]-peroxidase substrate(Kirkegaard & Perry Laboratories, Gaithersburg, Md.). OD values wereobtained using a Titertek® plate reader using a 405 nm filter andincluded into a computer spreadsheet. Antibody titers were expressed asthe serum dilutions that give OD=1.0.

Mouse IgG subclass production was analyzed using a similar assay butreplacing the secondary antibody with anti-mouse IgG1, IgG2a, IgG2b orIgG3 HRP-conjugated secondary antibody (Southern biotech)

Western Blot.

LSA-NRC(H)Mut, LSA-1 N term, LSA-1 C-term, AMA-1, TRAP, EBA-175 andAMA-1/E samples at 0.2-0.5 μg/lane were run on 4-12% Bis-Tris(Invitrogen, Carlsbad, Calif.) gel at 150 volts for 45 min, RT, andtransferred into nitrocellulose paper filters. The filters were blockedfor 1 hr with 1×PBST-5% casein (Sigma, St. Louis, Mo.), and washed fourtimes with PBST. Filters strips containing one lane with LSA-NRC(H)Mut,or several lanes with all the related or unrelated malaria antigens,mentioned above, were incubated with sera from individuals exposed to P.falciparum or with sera from immunized animals at 1:200 and 1:500dilutions. Incubation was allowed for 2 hr at RT then the filter werewashed and incubated with the respective anti-IgG AP-conjugatedsecondary antibody for another hr. Recognition of antigen by serumantibodies was noted after development with a solution containingNBT/BCIP substrate (Roche diagnostics, Indianapolis Ind.).

IFA:

The human hepatoblastoma cell line HC04 was grown on Lab-Tek® slidechambers (Nalge-Nunc, Rochester, N.Y.) and infected with NF54 P.falciparum sporozoites. After 7-8 days post-infection, cells were fixedwith cold methanol and incubated with preimmune and immune rabbit sera(post-2^(nd) immunization at 1:200) for 45 min at RT. Slides were washedwith 1×PBS and incubates during 45 min with goat anti-rabbit IgGFITC-conjugated secondary antibody (Southern biotech, Birmingham, Ala.)at 1:100 dilution. Recognition of native LSA-1 P. falciparum protein oninfected hepatocytes was visualized using a 492-nm UV microscopy.Preimmune rabbit serum and a mouse monoclonal antibody to the P.falciparum HSP-60 protein were used as negative and positive controlrespectively.

IFN-Gamma Production on Mice:

The Interferon-gamma detection module (R&D systems Inc. Minneapolis,Minn.) was used to determine the ex-vivo production of this cytokine inresponse to LSA-NRC(H)Mut. For this assay, spleen cells from LSA-NRC^(H)immunized C57BL/6 and C57BL/6 TgN mice, at 2.0×10⁵ cells per well, werecultured in Iscove's modified Dulhecco's medium supplemented with 10%fetal bovine serum (BioWhittaker, Walkersville, Md.), 2 mM L-glutamine,55 μM 2-mercaptoethanol, 1 mM sodium pyruvate, 0.1 mM nonessential aminoacids, and 100 U/ml of penicillin-streptomycin (Invitrogen) at 37° C.under a humidified atmosphere with 5% CO₂ for 48 hours. Cultures weredone in triplicate in the presence of LSA-NRC(H)Mut, the derived LSA-1peptides PL910 and PL911 (VSQTNFKSL (SEQ ID NO:27) and SQTNFKSL (SEQ IDNO:28), respectively) at 10 μg/ml or a mixture of both peptides, at 5μg/ml-each, in 96-well filtration plates (MultiScreen® HA, Millipore,Billerica, Mass.) previously coated with a mouse IFN-gamma capturemonoclonal antibody. Cultures done in presence of 10 μg/ml AMA-1/E, 5μg/ml concanavalin A, or medium alone were used as control. Plates werethen washed eight times with 1×PBS and incubated overnight at 4° C. witha mouse IFN-gamma detection/biotinylated monoclonal antibody. Plateswere washed eight times and were incubated at RT for 2 hr with astreptavidin-AP conjugate. Plates were washed ten times and developedwith NBT/BCIP. ELISPOTS were quantified manually using a dissectionmicroscope.

EXAMPLE 1

We have produced a recombinant product based on the LSA-1 protein fromP. falciparum 3D7 strain. Using codon harmonization, a novel approach,we have been able to enhance the expression of a gene lsa-nrc^(hmut),that encodes a protein LSA-NRC(H)Mut in E. coli Tuner (DE3) strain. Theexpressed recombinant protein is in soluble form even at high expressionlevels.

We have developed a three column chromatographic purification schemethat results in an LSA-NRC product that is >99% pure (see FIG. 1). Thepurification profiles of the protein through the steps is shown in FIG.2. The final amount of purified protein obtained is approximately 5 g/kgof starting bacterial paste. The final purification product was analyzedon an SDS-PAGE gel and silver stained (FIG. 3). The minor band whichstarts to appear in the 1.0 ug load has been determined, by N-terminalsequencing to be an LSA-NRC(H)Mut product that is the result of asecondary initiation site at the Met at position 62 in the protein.Scans of the gel show that it is less that 10% of the major band. Theminor band that appears above the major band starting in load 4.0 ug isa dimmer of LSA-NRC(H)Mut. Host cell protein analysis indicates lessthan 0.1 ug E. coli per 50 ug product (data not shown).

EXAMPLE 2

Two mouse strains, C57Blk/6 and Balb/c, have been immunized withLSA-NRC(H)Mut protein emulsified in Montanide 720 adjuvant. While theBalb/c mice responded to the protein by making antibodies, the C57Blk/7mice were nonresponders (FIG. 4). This is similar to observations ofJoshi et al. (2000, supra) who showed that C57Blk/6 mice were alsonon-responders to synthetic peptide epitopes of LSA-1. The major isotypeantibody formed in Balb/c mice was IgG1, with some IgG2a and IgG2bformed (FIG. 5). Very little IgG3 was made in Balb/c mice in response toLSA-NRC(H)Mut (FIG. 5).

Serum from rabbits immunized with LSA-NRC(H)Mut also containedantibodies that recognize the N-terminal and the C-terminal protions ofLSA-NRC(H)Mut (FIG. 7). Similarly, a Western blot of human serumobtained from adult individuals living in malaria endemic areas in Kenyashows that antibodies from infected humans recognize LSA-NRC(H)Mut (FIG.9).

EXAMPLE 3

The interferon-gamma detection module was used to determine the ex-vivoproduction of this cytokine in response to LSA-NRC(H)Mut. For thisassay, spleen cells from LSA-NRC(H)Mut immunized C57Blk/6 and C57BL/6TgN mice were cultured in the presence of LSA-NRC(H)Mut on filtrationplates previously coated with a mouse IFN-gamma capture monoclonalantibody. After washing, IFN-gamma detection/biotinylated monoclonalantibody was added and ELISPOTS were quantified. Results indicate thatspleen cells from mice immunized with LSA-NRC(H)Mut tended to produceIFN-gamma (FIG. 6).

EXAMPLE 4

Recognition of native LSA-1 P. falciparum protein on infectedhepatocytes was visualized using a 492-nm UV microscopy using serum fromLSA-NRC(H)Mut immunized rabbits. FIG. 8 shows that the immune serumrecognized a schizont developing in the infected hepatocyte.

1. A recombinant LSA-NRC polypeptide comprising at least one LSA-1epitope.
 2. A recombinant LSA-NRC polypeptide according to claim 1wherein said polypeptide comprises any of the epitopes defined by (i)codons encoding P. falciparum LSA-1 N-terminal; (ii) codons encoding P.falciparum LSA-1 C-terminal; (iii) one or more 17 amino acid repeat unitGluGlnGlnSerAspLeuGluGlnGluArgLeuAlaLysGluLysLeuGln (SEQ ID NO:1); (iv)one or more amino acid repeat unitGluGlnGlnArgAspLeuGluGlnGluArgLeuAlaLysGluLysLeuGln (SEQ ID NO:2); (v)one or more amino acid repeat unit following the order: X ¹ GlnGlnX ²AspX ³ GluGlnX ⁴ ArgX ⁵ AlaX ⁶ GluX ⁷ LueGln (SEQ ID NO:5) where x₁ iseither Glu or Gly; x₂ is Ser or Arg; x₃ is Asp or Ser; x₄ is Glu or Asp;x₅ is Leu or Arg; x₆ is Lys or Asn and x₇ is Lys or Thr or Arg; and (vi)one or more epitope specified in SEQ ID NO:6-23.
 3. A polypeptideaccording to claim 2 wherein said polypeptide is harmonized.
 4. Thepolypeptide of claim 3 wherein said polypeptide is LSA-NRC(H) specifiedin SEQ ID NO:26.
 5. The LSA-NRC(H) polypeptide according to claim 3further comprising a mutation in the T5 epitope, LSA-NRC(H)Mut,specified in SEQ ID NO:4.
 6. A composition comprising the recombinant P.falciparum LSA-NRC of claim
 1. 7. A composition comprising therecombinant polypeptide of claim
 2. 8. A composition comprising therecombinant polypeptide of claim
 3. 9. A composition comprising therecombinant polypeptide of claim
 4. 10. A composition comprising therecombinant polypeptide of claim
 5. 11. A recombinant vector comprisinga DNA sequence encoding LSA-NRC according to claim
 1. 12. A recombinantvector comprising a DNA sequence encoding LSA-NRC according to claim 2.13. A recombinant vector comprising a DNA sequence encoding LSA-NRC(H)according to claim
 4. 14. A recombinant vector comprising a DNA sequenceencoding LSA-NRC(H)Mut according to claim
 5. 15. A recombinant vectorcomprising a DNA sequence encoding LSA-NRC according to claim
 3. 16. Thevector of claim 12 wherein said DNA sequence corresponds to SEQ IDNO:25.
 17. The vector of claim 13 wherein said DNA sequence correspondsto SEQ ID NO:3.
 18. The vector of claim 16 wherein said vector ispETK(−).
 19. The vector of claim 17 wherein said vector is pETK(−). 20.The vector of claim 19 wherein said vector is pET KLSA-NRC^(hmut).
 21. Ahost cell transformed with the vector according to claim
 18. 22. A hostcell transformed with the vector according to claim
 20. 23. The hostcell of claim 20 wherein said host is E. coli Tuner (DE3).
 24. A methodfor producing and purifying recombinant P. falciparum LSA-NRCpolypeptide comprising: (i) growing a host cell containing a vectorexpressing P. falciparum LSA-NRC polypeptide in a suitable culturemedium, (ii) causing expression of said vector under suitable conditionsfor production of soluble LSA-NRC polypeptide and, (iii) lysing saidhost cells and recovering said LSA-NRC polypeptide such that it retainsits native folding.
 25. The method of claim 24 further comprisingremoval of E. coli endotoxin.
 26. The method of claim 25 wherein saidremoval of endotoxin is by (i)application of the lysed bacteria to aresin containing Ni-NTA and washing said resin bound material with lowpH, high salt buffer, (ii)removal of bound material from Ni-NTA resinand binding to other ion affinity resins such as DEAE and SP-Sepharoseresins such that the LSA-NRC polypeptide binds and the endotoxins can bewashed away.
 27. An antibody produced against the recombinant LSA-NRCpolypeptide of claim
 1. 28. An antibody produced against the recombinantLSA-NRC polypeptide of claim
 2. 29. An antibody produced against therecombinant LSA-NRC(H) polypeptide of claim
 4. 30. An antibody producedagainst the recombinant LSA-NRC(H)Mut polypeptide of claim
 5. 31. Anantibody produced against the recombinant LSA-NRC polypeptide of claim3.
 32. The antibody of claim 27 wherein said antibody is monoclonal orpolyclonal.
 33. The antibody of claim 28 wherein said antibody ismonoclonal or polyclonal.
 34. The antibody of claim 29 wherein saidantibody is monoclonal or polyclonal.
 35. The antibody of claim 30wherein said antibody is monoclonal or polyclonal.
 36. The antibody ofclaim 31 wherein said antibody is monoclonal or polyclonal.
 37. A methodfor in vitro diagnosis or detection of malaria antigen present in abiological sample, comprising: (i) contacting said biological samplewith a LSA-NRC specific antibody according to claim 27, preferably in animmobilized form under appropriate conditions which allow the formationof an immune complex, (ii) removing unbound components, (iii) incubatingthe immune complexes formed with heterologous antibodies whichspecifically bind to the antibodies present in the sample to beanalyzed, with said heterologous antibodies conjugated to a detectablelabel under appropriate conditions, (iv) detecting the presence of saidimmune complexes visually or mechanically.
 38. A kit for in vitrodetection of a malaria antigen present in a biological sample,comprising: (i)at least one antibody which reacts with recombinantLSA-NRC according to claim 27, said antibody being preferentiallyimmobilized on a solid substrate, (ii)a buffer, or components necessaryfor producing the buffer, enabling binding reaction between theseantibodies and the malaria antigens present in the biological sample,and (iii)a means for detecting the immune complexes formed in thepreceding binding reaction.
 39. A recombinant protein according to anyone of claims 1-5, wherein said purified protein is at least 90% pure.40. An immunogenic carrier comprising a polypeptide according toclaim
 1. 41. An immunogenic carrier comprising a polypeptide accordingto claim
 2. 42. An immunogenic carrier comprising a polypeptideaccording to claim
 4. 43. An immunogenic carrier comprising apolypeptide according to claim
 5. 44. An immunogenic carrier comprisinga polypeptide according to claim
 3. 45. A method for in vitro diagnosisof malaria antibodies in a biological sample, comprising (i) contactingsaid biological sample with a composition comprising a LSA-NRCpolypeptide according to claim 1 under appropriate conditions whichallow the formation of an immune complex, wherein said peptide islabeled with a detectable label, and (ii) detecting the presence of saidimmune complexes visually or mechanically.
 46. A kit for determining thepresence of malaria antibodies in a biological sample, comprising: (i)atleast one polypeptide or protein composition according to claim 9, abuffer or components necessary for producing a buffer; (ii) means fordetecting immune complexes formed between the peptide and antibodiespresent in the sample.
 47. A kit for determining the presence of malariaantibodies in a biological sample, comprising: (i)at least onepolypeptide or protein composition according to claim 10, a buffer orcomponents necessary for producing a buffer; (ii) means for detectingimmune complexes formed between the peptide and antibodies present inthe sample.
 48. A method for in vitro monitoring malaria infection orprognosing the response to treatment of patients suffering from malariainfection comprising: (i) incubating a biological sample from a patientwith malaria infection with an LSA-NRC protein according to claim 1 or asuitable part thereof under conditions allowing the formation of animmunological complex, (ii) removing unbound components, calculating theanti-LSA-1 titers present in said sample.
 49. A kit for monitoringmalaria infection or prognosing the response to treatment of patientssuffering from malaria infection comprising: (i)at least one LSA-NRCpeptide according to claim 1, (ii) a buffer or buffer components, (iii)means for detecting the immune complexes formed between the peptide andantibodies present in the sample, and (iv) optionally, a means fordetermining the amount of immune complex formed.
 50. An immunogeniccomposition comprising P. falciparum LSA-NRC of claim
 1. 51. Animmunogenic composition comprising the polypeptide according to claim 2.52. An immunogenic composition comprising the polypeptide according toclaim
 4. 53. An immunogenic composition comprising the polypeptideaccording to claim
 5. 54. An immunogenic composition comprising thepolypeptide according to claim
 3. 55. The immunogenic composition ofclaim 50 further comprising an adjuvant.
 56. The immunogenic compositionof claim 51 further comprising an adjuvant.
 57. The immunogeniccomposition of claim 52 further comprising an adjuvant.
 58. Theimmunogenic composition of claim 53 further comprising an adjuvant. 59.The immunogenic composition of claim 54 further comprising an adjuvant.60. The immunogenic composition of claim 55 wherein said adjuvant ischosen from the group consisting of: Montanide and alum.
 61. Theimmunogenic composition of claim 56 wherein said adjuvant is chosen fromthe group consisting of: Montanide and alum.
 62. The immunogeniccomposition of claim 57 wherein said adjuvant is chosen from the groupconsisting of: Montanide and alum.
 63. The immunogenic composition ofclaim 58 wherein said adjuvant is chosen from the group consisting of:Montanide and alum.
 64. The immunogenic composition of claim 59 whereinsaid adjuvant is chosen from the group consisting of: Montanide andalum.
 65. A method for inducing in a subject an immune response againstmalaria infection comprising administering to said subject a compositioncomprising an immunologically effective amount of P. falciparum LSA-NRCof claim 1 in an acceptable diluent.
 66. The method of claim 65 whereinsaid composition further comprises an adjuvant.
 67. The composition ofclaim 66 wherein said adjuvant is selected from the group consisting ofMontanide, and alum.
 68. A method for inducing in a subject an immuneresponse against malaria infection comprising administering to saidsubject a composition comprising an immunologically effective amount ofP. falciparum LSA-NRC of claim 2 in an acceptable diluent.
 69. Themethod of claim 68 wherein said composition further comprises anadjuvant.
 70. The composition of claim 69 wherein said adjuvant isselected from the group consisting of Montanide, and alum.
 71. A methodfor inducing in a subject an immune response against malaria infectioncomprising administering to said subject a composition comprising animmunologically effective amount of P. falciparum LSA-NRC of claim 3 inan acceptable diluent.
 72. The method of claim 71 wherein saidcomposition further comprises an adjuvant.
 73. The composition of claim72 wherein said adjuvant is selected from the group consisting ofMontanide, and alum.
 74. A method for inducing in a subject an immuneresponse against malaria infection comprising administering to saidsubject a composition comprising an immunologically effective amount ofP. falciparum LSA-NRC of claim 4 in an acceptable diluent.
 75. Themethod of claim 74 wherein said composition further comprises anadjuvant.
 76. The composition of claim 75 wherein said adjuvant isselected from the group consisting of Montanide, and alum.
 77. A methodfor inducing in a subject an immune response against malaria infectioncomprising administering to said subject a composition comprising animmunologically effective amount of P. falciparum LSA-NRC of claim 5 inan acceptable diluent.
 78. The method of claim 77 wherein saidcomposition further comprises an adjuvant.
 79. The composition of claim78 wherein said adjuvant is selected from the group consisting ofMontanide, and alum.
 80. A method for inducing a protective immuneresponse to malaria in a mammal, comprising administering a compositioncomprising a P. falciparum LSA-NRC according to claim 1 in an amounteffective to induce an immune response in said mammal.
 81. The methodaccording to claim 80 wherein the composition further comprises anadjuvant selected from the group consisting of Montanide, and alum. 82.A method for inducing a protective immune response to malaria in amammal, comprising administering a composition comprising a P.falciparum LSA-NRC according to claim 2 in an amount effective to inducean immune response in said mammal.
 83. The method according to claim 82wherein the composition further comprises an adjuvant selected from thegroup consisting of Montanide, and alum.
 84. A method for inducing aprotective immune response to malaria in a mammal, comprisingadministering a composition comprising a P. falciparum LSA-NRC accordingto claim 3 in an amount effective to induce an immune response in saidmammal.
 85. The method according to claim 84 wherein the compositionfurther comprises an adjuvant selected from the group consisting ofMontanide, and alum.
 86. A method for inducing a protective immuneresponse to malaria in a mammal, comprising administering a compositioncomprising a P. falciparum LSA-NRC according to claim 4 in an amounteffective to induce an immune response in said mammal.
 87. The methodaccording to claim 86 wherein the composition further comprises anadjuvant selected from the group consisting of Montanide, and alum. 88.The method according to claim 84 wherein the composition furthercomprises an adjuvant selected from the group consisting of Montanide,and alum.
 89. A method for inducing a protective immune response tomalaria in a mammal, comprising administering a composition comprising aP. falciparum LSA-NRC according to claim 5 in an amount effective toinduce an immune response in said mammal.
 90. The method according toclaim 89 wherein the composition further comprises an adjuvant selectedfrom the group consisting of Montanide, and alum.
 91. A multivalentvaccine for protection against infection with more than one strain of P.falciparum, said vaccine comprising LSA-NRC polypeptides from more thanone strain of P. falciparum chosen from the group consisting of: 3D7,FVO, T9/96, NF54, and (what about other strains) camp.
 92. Themultivalent vaccine of claim 91, further comprising an adjuvant selectedfrom the group consisting of Montanide, and alum.